DK2568049T3 - Mice expressing a hybrid immunoglobulin light chain with a human variable region - Google Patents

Description

DESCRIPTION

FIELD

[0001] Genetically modified mice that comprise an unrearranged human A immunoglobulin light chain variable gene segment (hVA) and a human λ joining gene segment (hJA) operably linked with an intact mouse lambda (A) light chain constant region. Genetically modified mice that express antibodies that comprise an immunoglobulin light chain derived from a hVA, a hJA and a mouse Cλ gene.

BACKGROUND

[0002] Mice that express antibodies that are fully human, or partly human and partly mouse, are known in the art. For example, transgenic mice that express fully human antibodies from transgenes containing human light and heavy chain immunoglobulin variable region genes have been reported. Genetically modified mice that comprise a replacement of the endogenous mouse heavy chain variable region (HCVR) gene segments and kappa (k) light chain variable region (LCVR) gene segments with human HCVR and LCVR gene segments and that make chimeric antibodies with a chimeric human/mouse kappa chain are known as well.

[0003] Antibody light chains are encoded by one of two separate loci: kappa (k) and lambda (A). Mouse antibody light chains are primarily of the κ type. The ratio of κ to λ light chain usage in humans is about 60:40, whereas in mice it is about 95:5. Biased usage of κ light chains in mice is reportedly sustained in genetically modified mice capable of expressing fully or partly human antibodies. Thus, mice that express fully or partly human antibodies appear to be constrained in lambda variable usage.

[0004] There is a need in the art to generate lambda variable regions, whether mouse or human, for use in making epitopebinding proteins. There is a need in the art for mice that express fully or partly human antibodies, wherein the mice display an increased lambda variable (VA) usage.

[0005] There is a need in the art for mice that express fully or partly human antibodies, wherein the mice display an increased λ variable (VA) usage. WO 00/26373 refers to murine expression of human Ig A locus.

[0006] WO 2010/039900 relates to knock-in non-human cells and mammals having a genome encoding chimeric Ig chains and antibodies and methods for producing such knock-in cells and mammals. A knock-in mouse having a genome comprising a human VH segment, a human JH gene segment and a human DH gene segment and a portion of a syngeneic Ig heavy chain locus, is disclosed, where the human heavy chain locus replaces all or portion of an endogenous heavy chain locus so that the animal is capable of producing chimeric heavy chains. In specific embodiments the mouse genome further comprises a human IgA light chain locus, or a portion thereof, and an IgA 3'LCR, where the human IgA light chain locus replaces all or portion of an endogenous Ig light chain locus.

SUMMARY

[0007] The invention in its broadest sense is as defined in the independent claims. Genetically modified mice, embryos, cells, tissues, as well as nucleic acid constructs for modifying mice, and methods and compositions for making and using them, are disclosed. Mice and cells that generate human A variable regions in the context of a A light chain, from an endogenous mouse light chain locus, are also provided as recited in the claims. Also disclosed are methods for making antibodies that comprise lambda variable regions. Methods for selecting heavy chains that express with cognate lambda variable regions are also disclosed.

[0008] Chimeric and human antigen-binding proteins (e.g., antibodies), and nucleic acids encoding them, are disclosed that comprise somatically mutated variable regions, including antibodies that have light chains comprising a variable domain derived from a human VA and a human JA gene segment fused to a mouse lambda light chain constant domain.

[0009] The present invention provides a mouse comprising: 1. (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and 2. (b) an endogenous A (lambda) light chain allele comprising an unrearranged human lambda variable (hVA) and an unrearranged human lambda joining (hJA) gene segment, where the mouse comprises a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse lambda constant (CA) region gene of said second VA-JA-CA gene cluster so the mouse expresses a light chain derived from a hVA, a hJA and a mouse CA gene, where the endogenous enhancers Enh 2.4, mouse lambda 3'enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele.

[0010] The invention further provides: an isolated cell that expresses a light chain derived from a hVA, a hJA and a mouse CA gene, wherein the cell is from, or obtainable from, the mouse of any one of the preceding claims, and wherein the cell comprises: (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse CA constant region gene of said second VA-JA-CA gene cluster and wherein the endogenous enhancers Enh 2.4, mouse lambda 3' enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele; an isolated mouse embryonic stem (ES) cell comprising: (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising an unrearranged human lambda variable (hVA) and an unrearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and a replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse CA constant region gene of said second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3' enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele; a mouse embryo comprising, made from, or obtainable from, the mouse ES cell of the invention, wherein the mouse embryo comprises: (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising an unrearranged human lambda variable (hVA) and an unrearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and a replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse CA constant region gene of said second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3' enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele; a hybridoma that expresses a light chain derived from a hVA, a hJA and a mouse CA gene wherein the hybridoma is from, or obtainable from, the mouse of the invention, wherein the cell comprises: (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse CA constant region gene of said second VA-JA-CA gene cluster and wherein the endogenous enhancers Enh 2.4, mouse lambda 3' enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele; and use of a B cell of the invention to make a hybridoma that expresses a light chain derived from a hVA, a hJA and a mouse CA gene, wherein the hybridoma is from, or obtainable from, the mouse of the invention, wherein the cell comprises: (a) a replacement of one or more heavy chain variable (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh, and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster of said endogenous mouse A light chain allele and replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of said allele with said human VA and JA gene segments which are operably linked to an intact mouse CA constant region gene of said second VA-JA-CA gene cluster and wherein the endogenous enhancers Enh 2.4, mouse lambda 3' enhancer (Enh) and Enh 3.1 are maintained intact in said A light chain allele; [0011] The present invention further provides a method of making an antibody in a mouse comprising: (a) exposing a mouse of the invention to an antigen; (b) allowing the mouse to develop an immune response to the antigen; and (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, or identifying in the mouse of (b) a nucleic acid sequence encoding a heavy and/or light chain variable domain that binds the antigen, wherein the antibody comprises a light chain derived from a hVA, a hJA and a mouse CA gene.

[0012] Antibodies are disclosed that comprise (a) a human heavy chain variable domain (hV|-|) fused to a mouse heavy chain constant region, and (b) a human VA fused to a mouse Cl domain; including wherein one or more of the variable domains are somatically mutated, e.g., during antibody or immune cell selection in a mouse of the invention. The unrearranged hVA and unrearranged hJA are operably linked with an intact mouse A constant region (CA).

[0013] Also disclosed is a mouse that comprises in its germline, at an endogenous mouse light chain locus, a human A light chain variable region sequence, wherein the human lambda variable region sequence is expressed in a light chain that comprises a mouse immunoglobulin lambda constant region gene sequence.

[0014] The endogenous mouse light chain locus is a A locus.

[0015] In one aspect, the mouse lacks an endogenous light chain variable sequence at the endogenous mouse light chain locus.

[0016] In one aspect, all or substantially all endogenous mouse light chain variable region gene segments are replaced with one or more human A variable region gene segments.

[0017] The human A light chain variable region sequence comprises a human JA sequence. In one aspect, the human JA sequence is selected from the group consisting of JA1, JA2, JA3, JA7, and a combination thereof.

[0018] In one aspect, the human A light chain variable region sequence comprises a fragment of cluster A of the human light chain locus. In a specific aspect, the fragment of cluster A of the human A light chain locus extends from hVA3-27 through hVA3-1.

[0019] In one aspect, the human A light chain variable region sequence comprises a fragment of cluster B of the human light chain locus. In a specific aspect, the fragment of cluster B of the human A light chain locus extends from hVA5-52 through hVA1-40.

[0020] In one aspect, the human A light chain variable region sequence comprises a genomic fragment of cluster A and a genomic fragment of cluster B. In a one aspect, the human A light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B.

[0021] In one aspect, more than 10% of the light chain naive repertoire of the mouse is derived from at least two hVA gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. In one aspect, more than 20% of the light chain naive repertoire of the mouse is derived from at least three hVA gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49. In one aspect, more than 30% of the light chain naive repertoire of the mouse is derived from at least four hVA gene segments selected from 2-8, 2-23, 1-40, 5-45, and 9-49.

[0022] [Deleted] [0023] [Deleted] [0024] [Deleted] [0025] [Deleted] [0026] [Deleted] [0027] [Deleted] [0028] [Deleted] [0029] [Deleted] [0030] [Deleted] [0031] [Deleted] [0032] [Deleted] [0033] [Deleted] [0034] [Deleted] [0035] [Deleted] [0036] [Deleted] [0037] [Deleted] [0038] Also disclosed is a genetically modified mouse, wherein the mouse comprises an unrearranged immunoglobulin VA and a Jλ gene segment operably linked to a mouse light chain locus that comprises an intact mouse CA gene.

[0039] The VA and JA gene segments are human gene segments.

[0040] The endogenous mouse light chain locus is a A light chain locus.

[0041] [Deleted] [0042] [Deleted] [0043] The mouse further comprises a replacement of one or more heavy chain V, D, and/or J gene segments with one or more human V, D, and/or J gene segments at an endogenous mouse heavy chain immunoglobulin locus.

[0044] [Deleted] [0045] In one aspect, the mouse comprises an unrearranged human immunoglobulin A light chain variable gene segment (VA) and a A joining gene segment (JA) at an endogenous mouse A light chain locus that comprises an intact mouse CA gene.

[0046] [Deleted] [0047] [Deleted] [0048] The A light chain variable gene locus (the "A locus") comprises at least one hVA gene segment and at least one human JA (hJA) gene segment. In another aspect, the A locus comprises up to four hJA gene segments.

[0049] In one aspect, the VA locus comprises a plurality of hVAs. In one aspect, the plurality of hVAs are selected so as to result in expression of a A light chain variable region repertoire that reflects about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the VA usage observed in a human. In one aspect, the VA locus comprises gene segments hVA 1-40, 1-44, 2-8, 2-14, 3-21, and a combination thereof.

[0050] In one aspect, the hVAs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific aspect, the VA locus comprises a contiguous sequence of the human A light chain locus that spans from VA3-12 to VA3-1. In one aspect, the VA locus comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVAs. In a specific aspect, the hVAs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific aspect, the VA locus comprises a contiguous sequence of the human A locus that spans from VA3-12 to VA3-1. In a specific aspect the endogenous κ locus is deleted in part or completely.

[0051] In one aspect, the VA locus comprises 13 to 28 or more hVAs. In a specific aspect, the hVAs include 2-14, 3-16, 2-18, 3- 19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a specific aspect the endogenous κ locus is deleted in part or completely.

[0052] In one aspect, the VA locus comprises 29 to 40 hVAs. In a specific embodiment, all or substantially all sequence between hVA1-40 and hVA3-29 in the genetically modified mouse consists essentially of a human A sequence of approximately 959 bp found in nature (e.g., in the human population) downstream of the hVA1-40 gene segment (downstream of the 3' untranslated portion), a restriction enzyme site (e.g., Pl-Scel), followed by a human A sequence of approximately 3,431 bp upstream of the hVA3-29 gene segment found in nature. In a specific aspect, the endogenous mouse κ locus is deleted in part or completely.

[0053] The VA locus comprises at least one hJA. In one aspect, the VA locus comprises a plurality of hJAs. In one aspect, the VA locus comprises at least 2, 3, 4, 5, 6, or 7 hJA. In a specific aspect, the VA locus comprises four hJA. In a specific aspect, the four hJAs are hJA1, hJA2, hJA3, and hJA7. In one aspect, the VA locus comprises one hJA. In a specific aspect, the one hJA is hJA1. In a specific aspect, the endogenous κ locus is deleted in part or completely.

[0054] The VA locus comprises at least one hVA, at least one hJA, and an intact mouse CA gene. In a specific aspect, the intact mouse CA gene is CA2.

[0055] [Deleted] [0056] In one aspect, the mouse comprises a replacement of endogenous mouse VA gene segments at the endogenous mouse A locus with one or more human VA gene segments at the endogenous mouse A locus, wherein the hVA gene segments are operably linked to an intact mouse CA region gene, such that the mouse rearranges the hVA gene segments and expresses a reverse chimeric immunoglobulin light chain that comprises a hVA domain and a mouse CA. In a specific aspect, the intact mouse CA gene is CA2. In one aspect, 90-100% of unrearranged mouse VA gene segments are replaced with at least one unrearranged hVA gene segment. In a specific aspect, all or substantially all of the endogenous mouse VA gene segments are replaced with at least one unrearranged hVA gene segment. In one aspect, the replacement is with at least 12, at least 28, or at least 40 unrearranged hVA gene segments. In one embodiment, the replacement is with at least 7 functional unrearranged hVA gene segments, at least 16 functional unrearranged hVA gene segments, or at least 27 functional unrearranged hVA gene segments. In one embodiment, the mouse comprises a replacement of all mouse JA gene segments with at least one unrearranged hJA gene segment. In one aspect, the at least one unrearranged hJA gene segment is selected from JA1, JA2, JA3, JA4, JA5, JA6, JA7, and a combination thereof. In a specific embodiment, the one or more hVA gene segment is selected from a 3-1,4-3, 2-8, 3-9, 3-10, 2-11, 3-12, 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, 3-27, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 5-48, 9-49, 1-50, 1-51, a 5-52 hVA gene segment, and a combination thereof. In a specific aspect, the at least one unrearranged hJA gene segment is selected from JA1, JA2, JA3, JA7, and a combination thereof.

[0057] [Deleted] [0058] [Deleted] [0059] [Deleted] [0060] [Deleted] [0061] [Deleted] [0062] [Deleted] [0063] Also disclosed is a genetically modified mouse, wherein the mouse comprises no more than two light chain alleles, wherein the light chain alleles comprise (a) an unrearranged immunoglobulin human VA and a JA gene segment at an endogenous mouse light chain locus that comprises an intact mouse CA gene; and, (b) an unrearranged immunoglobulin V|_ and a J|_ gene segment at an endogenous mouse light chain locus that comprises a mouse Cl gene.

[0064] In one aspect, the endogenous mouse light chain locus is a κ locus. In another aspect, the endogenous mouse light chain locus is a A locus.

[0065] In one aspect, the no more than two light chain alleles are selected from a κ allele and a A allele, and two A alleles. In a specific aspect, one of the two light chain alleles is a λ allele that comprises an intact CA2 gene.

[0066] [Deleted] [0067] In one aspect, the mouse comprises one functional immunoglobulin light chain locus and one nonfunctional light chain locus, wherein the functional light chain locus comprises an unrearranged immunoglobulin human VA and a Jλ gene segment at an endogenous mouse λ light chain locus that comprises an intact mouse Cλ gene. In one aspect, the intact Cλ gene is CA2.

[0068] The mouse further comprises at least one immunoglobulin heavy chain allele. In one aspect, the at least one immunoglobulin heavy chain allele comprises a human Vh gene segment, a human Dh gene segment, and a human Jh gene segment at an endogenous mouse heavy chain locus that comprises a human heavy chain gene that expresses a human/mouse heavy chain. In a specific aspect, the mouse comprises two immunoglobulin heavy chain alleles, and the mouse expresses a human/mouse heavy chain.

[0069] [Deleted] [0070] In one aspect, the mouse comprises a first light chain allele that comprises an unrearranged hVK and an unrearranged hJK, at an endogenous mouse κ locus that comprises an endogenous Ck gene; and a second light chain allele that comprises an unrearranged hVA and an unrearranged hJA, at an endogenous mouse λ locus that comprises an intact endogenous Cλ gene as recited in the claims. In a specific aspect, the first and the second light chain alleles are the only functional light chain alleles of the genetically modified mouse. In one aspect, the intact endogenous Cλ gene is CA2.

[0071] In one aspect, the mouse comprises six immunoglobulin alleles, wherein the first allele comprises an unrearranged immunoglobulin VA and JA gene segment at an endogenous mouse κ light chain locus that comprises a mouse Ck gene, the second comprises an unrearranged immunoglobulin Vk and Jk gene segment at an endogenous mouse κ light chain locus that comprises a mouse Ck gene, the third comprises an unrearranged immunoglobulin VA and JA gene segment at an endogenous mouse A light chain locus that comprises an intact mouse CA gene as recited in the claims, the fourth and fifth each independently comprise an unrearranged Vh and Dh and Jh gene segment at an endogenous mouse heavy chain locus that comprises a mouse heavy chain gene, and the sixth comprises either (a) an unrearranged immunoglobulin VA and JA gene segment at an endogenous mouse A light chain locus that comprises a mouse CA gene, (b) a A locus that is nonfunctional, or (c) a deletion in whole or in part of the A locus.

[0072] In one aspect, the first allele comprises an unrearranged hVA and hJA. In one aspect, the second allele comprises an unrearranged hVK and hJK. The third allele comprises an unrearranged hVA and hJA. In one aspect, the fourth and fifth each independently comprise an unrearranged hVn and hDn and hJH- In one aspect, the sixth allele comprises an endogenous mouse A locus that is deleted in whole or in part.

[0073] In one aspect, the mouse comprises six immunoglobulin alleles, wherein the first allele comprises an unrearranged immunoglobulin VA and JA gene segment at an endogenous mouse A light chain locus that comprises an intact mouse CA gene, the second comprises an unrearranged immumoglobulin VA and JA gene segment at an endogenous mouse A light chain locus that comprises a mouse CA gene, the third comprises an unrearranged immunoglobulin Vk and Jk gene segment at an endogenous mouse κ light chain locus that comprises a mouse Ck gene, the fourth and fifth each independently comprise an unrearranged Vh and Dh and Jh gene segment at an endogenous mouse heavy chain locus that comprises a mouse heavy chain gene, and the sixth comprises either (a) an unrearranged immunoglobulin Vk and Jk gene segment at an endogenous mouse κ light chain locus that comprises a mouse Ck gene, (b) a κ locus that is nonfunctional, or (c) a deletion of one or more elements of the κ locus.

[0074] In one aspect, the first allele comprises an unrearranged hVA and hJA gene segment. In one aspect, the second allele comprises an unrearranged hVA and hJA gene segment. In one aspect, the third allele comprises an unrearranged hVK and hJK gene segment. In one aspect, the fourth and fifth each independently comprise an unrearranged hVn and hDn and hJn gene segment. In one aspect, the sixth allele comprises an endogenous mouse κ locus that is functionally silenced.

[0075] In one aspect, the genetically modified mouse comprises a B cell that comprises a rearranged antibody gene comprising a rearranged hVA domain operably linked to an intact mouse CA domain. In a specific embodiment, the mouse CA domain is derived from a CA2 gene.

[0076] Also disclosed is a genetically modified mouse that expresses an antibody comprising a light chain that comprises a rearranged human VA-JA sequence and a mouse CA sequence.

[0077] [Deleted] [0078] [Deleted] [0079] [Deleted] [0080] [Deleted] [0081] [Deleted] [0082] [Deleted] [0083] [Deleted] [0084] [Deleted] [0085] Also disclosed is a mouse that expresses an antibody comprising a light chain derived from a hVA, a hJA and a mouse CA gene. In a specific aspect, the CA region is CA2. In a specific aspect the antibody further comprises a heavy chain comprising a variable domain derived from a human V, a human D and a human J gene segment, and a heavy chain constant domain derived from a mouse heavy chain constant region gene. In one aspect, the mouse heavy chain constant region gene comprises a hinge-CH2-CH3 sequence of a heavy chain constant domain. In another aspect, the mouse heavy chain constant region gene comprises a CH-|-hinge-CH2-CH3 sequence of a heavy chain constant domain. In another aspect, the mouse heavy chain constant region gene comprises a CH-1-CH2-CH3-CH4 sequence of a heavy chain constant domain. In another aspect, the mouse heavy chain constant region gene comprises a CH2-CH3-CH4 sequence of a heavy chain constant domain.

[0086] [Deleted] [0087] In one aspect, the mouse expresses an antibody comprising a light chain that comprises a rearranged immunoglobulin A light chain variable region comprising a human VA/JA sequence selected from 3-1/1, 3-1/7, 4-3/1, 4-3/7, 2-8/1, 3-9/1, 3-10/1, 3-10/3, 3-10/7, 2-14/1,3-19/1,2-23/1,3-25/1, 1-40/1, 1-40/2, 1-40/3, 1-40/7, 7-43/1,7-43/3, 1-44/1, 1-44/7, 5-45/1, 5-45/2, 5-45/7, 7-46/1,7-46/2, 7-46/7, 9-49/1, 9-49/2, 9-49/7 and 1-51/1.

[0088] Also disclosed is a mouse that expresses an antibody comprising (a) a heavy chain comprising a heavy chain variable domain derived from an unrearranged human heavy chain variable region gene segment, wherein the heavy chain variable domain is fused to a mouse heavy chain constant (Ch) region; and, (b) a light chain comprising a light chain variable domain derived from an unrearranged hVA and a hJA, wherein the light chain variable domain is fused to an intact mouse CA region.

[0089] [Deleted] [0090] In one aspect, the mouse comprises (i) a heavy chain locus that comprises a replacement of all or substantially all functional endogenous mouse V, D and J gene segments with all or substantially all functional human V, D, and J gene segments, a mouse Ch gene, (ii) a first A light chain locus comprising a replacement of all or substantially all functional endogenous mouse VA and JA gene segments with all, substantially all, or a plurality of, functional hVA and hJA gene segments, and an intact mouse CA gene, (iii) a second A light chain locus comprising a replacement of all or substantially all functional endogenous mouse VA and JA gene segments with all, substantially all, or a plurality of, functional hVA and hJA gene segments, and an intact mouse CA gene. In a specific aspect, the intact mouse CA gene is CA2.

[0091] In one aspect, the mouse comprises a deletion of a Ck gene and/or a Vk and/or a Jk gene segment. In one embodiment, the mouse comprises a nonfunctional κ light chain locus.

[0092] Also disclosed is a genetically modified mouse that expresses an antibody, wherein greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of total IgG antibody produced by the mouse comprises a λ-derived variable domain, and wherein the mouse expresses antibodies comprising a κ-derived variable domain fused with a mouse Ck region. In specific aspects, about 15-40%, 20-40%, 25-40%, 30-40%, or 35-40% of total antibody produced by the mouse comprises a A-derived variable domain.

[0093] In one aspect, the λ-derived variable domain is derived from a hN/λ and a hJA. In a specific aspect, the λ-derived variable region is in a light chain that comprises a mouse CK region. In another specific aspect, the CK region is a CK2 region. Also disclosed is an isolated DNA construct that comprises an upstream homology arm and a downstream homology arm, wherein the upstream and the downstream homology arms target the construct to a mouse κ locus, and the construct comprises a functional unrearranged hN/λ segment and a functional unrearranged Ιπύλ segment, and a selection or marker sequence.

[0094] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting a mouse λ sequence upstream of mouse \M2, a selection cassette flanked 5' and 3' with recombinase recognition sites, and a targeting arm for targeting a mouse λ sequence 3' of mouse JK2. In one aspect, the selection cassette is a Frt'ed Hyg-TK cassette. In one aspect, the 3' targeting arm comprises mouse Ολ2, ύλ4, Ολ4, and mouse enhancer 2.4.

[0095] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to \A1, a selection cassette flanked 5' and 3' with recombinase recognition sites, and a 3' targeting arm for targeting a mouse λ sequence 3' with respect to mouse Ολ1. In one aspect, the selection cassette is a loxed neomycin cassette. In one aspect, the 3' targeting arm comprises the mouse λ 3' enhancer and mouse λ 3' enhancer 3.1.

[0096] Also disclosed is an isolated DNA construct, comprising from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to VK2, a selection cassette flanked 5' and 3' with recombinase recognition sites, and a 3' targeting arm for targeting a mouse λ sequence 3' with respect to mouse JK2 and 5' with respect to mouse CK2. In one aspect, the selection cassette is a Frt'ed hygromycin-TK cassette. In one aspect, the 3' targeting arm comprises the mouse Ολ2-3λ4-Ολ4 gene segments and mouse λ enhancer 2.4.

[0097] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to VK2, a selection cassette flanked 5' and 3' with recombinase recognition sites, a human genomic fragment comprising a contiguous region of the human λ light chain locus from h\A3-12 downstream to the end of h^1, and a 3' targeting arm for targeting a mouse λ sequence 3' with respect to mouse ύλ2. In one aspect, the selection cassette is a Frt'ed neomycin cassette. In one aspect, the 3' targeting arm comprises the mouse Ολ2-3λ4-Ολ4 gene segments and mouse λ enhancer 2.4.

[0098] Also disclosed is an isolated DNA construct, comprising a contiguous region of the human λ light chain locus from h\A3-12 downstream to the end of h^1.

[0099] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to VK2, a selection cassette flanked 5' and 3' with recombinase recognition sites and a human genomic fragment comprising a contiguous region of the human λ light chain locus from h\A3-27 downstream to the end of h\A2-8. In one aspect, the selection cassette is a Frt'ed hygromycin cassette. In one aspect, the human genomic fragment comprises a 3' targeting arm. In a specific aspect, the 3' targeting arm comprises about 53 kb of the human λ light chain locus from h\A3-12 downstream to the end of h\A2-8.

[0100] Also disclosed is an isolated DNA construct, comprising a contiguous region of the human λ light chain locus from h\A3-27 downstream to the end of h\A3-12.

[0101] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse λ locus 5' with respect to VK2, a selection cassette flanked 5' and 3' with recombinase recognition sites, a first human genomic fragment comprising a contiguous region of the human λ light chain locus from h\A5-52 downstream to the end of h\A1-40, a restriction enzyme site, and a second human genomic fragment comprising a contiguous region of the human λ light chain locus from h\A3-29 downstream to the end of h\A82K. In one aspect, the selection cassette is a Frt'ed neomycin cassette. In one aspect, the restriction enzyme site is a site for a homing endonuclease. In a specific aspect, the homing endonuclease is Pl-Scel. In on aspect, the second human genomic fragment is a 3' targeting arm. In a specific aspect, the 3' targeting arm comprises about 27 kb of the human λ light chain locus from Ιιλ3-29 downstream to the end of h\A82K.

[0102] Also disclosed is an isolated DNA construct, comprising a contiguous region of the human λ light chain locus from h\A5-52 downstream to the end of h\A1-40.

[0103] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse κ locus 5' with respect to the endogenous Vk gene segments, two juxtaposed recombinase recognition sites, a selection cassette 3' to the juxtaposed recombinase recognition sites, and a 3' targeting arm for targeting a mouse κ sequence 5' with respect to the κ light chain variable gene segments. In one aspect, the juxtaposed recombinase recognition sites are in opposite orientation with respect to one another. In a specific aspect, the recombinase recognition sites are different. In another specific aspect, the recombinase recognition sites are a loxP site and a /ox511 site. In one aspect, the selection cassette is a neomycin cassette.

[0104] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a targeting arm for targeting the mouse κ locus 5' with respect to the mouse Jk gene segments, a selection cassette, a recombinase recognition site 3' to the selection cassette, and a 3' targeting arm for targeting a mouse κ sequence 3' with respect to the mouse Jk gene segments and 5' to the mouse κ intronic enhancer. In one aspect, the selection cassette is a hygromycin-TK cassette. In one aspect, the recombinase recognition site is in the same direction with respect to transcription as the selection cassette. In a specific aspect, the recombinase recognition site is a loxP site.

[0105] Also disclosed is an isolated DNA construct, comprising, from 5' to 3' with respect to the direction of transcription, a first mouse genomic fragment comprising sequence 5' of the endogenous mouse Vk gene segments, a first recombinase recognition site, a second recombinase recognition site, and a second mouse genomic fragment comprising sequence 3' of the endogenous mouse Jk gene segments and 5' of the mouse κ intronic enhancer.

[0106] Also disclosed is a genetically modified mouse, wherein the genetic modification comprises a modification with one or more of the DNA constructs described above or herein.

[0107] Also disclosed is the use of an isolated DNA construct to make a mouse as described herein. Also disclosed is the use of an isolated DNA construct as described herein in a method for making an antigen-binding protein.

[0108] Also disclosed is a non-human stem cell that comprises a targeting vector that comprises a DNA construct as described above and herein. Also disclosed is a non-human stem cell, wherein the non-human stem cell is derived from a mouse described herein.

[0109] In one aspect, the non-human stem cell is an embryonic stem (ES) cell. In a specific aspect, the ES cell is a mouse ES cell.

[0110] Also disclosed is the use of a non-human stem cell as described herein to make a mouse as described herein. Also disclosed is the use of a non-human stem cell as described herein to make an antigen-binding protein.

[0111] Also disclosed is a mouse embryo, wherein the mouse embryo comprises a genetic modification as disclosed herein. In one aspect, a host mouse embryo that comprises a donor ES cell is disclosed, wherein the donor ES cell comprises a genetic modification as described herein. In one aspect, the mouse embryo is a pre-morula stage embryo. In a specific aspect, the premorula stage embryo is a 4-cell stage embryo or an 8-cell stage embryo. In another specific aspect, the mouse embryo is a blastocyst.

[0112] Also disclosed is the use of a mouse embryo as described herein to make a mouse as described herein. In one aspect, use of a mouse embryo as described herein to make an antigen-binding protein is disclosed.

[0113] Also disclosed is a non-human cell, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one aspect, the cell is a B cell. In one aspect, the cell is a hybridoma. In one aspect, the cell encodes an immunoglobulin light chain variable domain and/or an immunoglobulin heavy chain variable domain that is somatically mutated.

[0114] Also disclosed is a non-human cell, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one aspect, the cell is a B cell. In one aspect, the cell is a hybridoma. In one aspect, the cell encodes an immunoglobulin light chain variable domain and/or an immunoglobulin heavy chain variable domain that is somatically mutated.

[0115] Also disclosed is the use of a non-human cell as described herein to make a mouse as described herein. In one aspect, use of a non-human cell as described herein to make an antigen-binding protein is disclosed.

[0116] Also disclosed is a mouse B cell that expresses an immunoglobulin light chain that comprises (a) a variable region derived from a hVA gene segment and a hJA gene segment; and, (b) a mouse CA gene. In a specific aspect, the CA gene is CA2. In one aspect, the mouse B cell further expresses a cognate heavy chain that comprises (c) a variable region derived from a hV|-|, a hDn, and (d) a hJn segment. In one aspect, the B cell does not comprise a rearranged A gene. In another aspect, the B cell does not comprise a rearranged κ gene.

[0117] Also disclosed is a method for making an antibody in a genetically modified mouse, comprising: (a) exposing a genetically modified mouse to an antigen, wherein the mouse has a genome comprising at least one hVA and at least one hJA at an endogenous light chain locus, wherein the endogenous light chain locus comprises a mouse CA gene; (b) allowing the genetically modified mouse to develop an immune response to the antigen; and, (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, wherein the antibody comprises a light chain derived from a hVA, a hJA and a mouse CA gene.

[0118] [Deleted] [0119] [Deleted] [0120] In one aspect, a method for making an antibody in a genetically modified mouse is disclosed, comprising: (a) exposing a genetically modified mouse to an antigen, wherein the mouse has a genome comprising at least one hVA at a λ light chain locus and at least one Jλ at the A light chain locus, wherein the A light chain locus comprises an intact mouse Cλ gene; (b) allowing the genetically modified mouse to develop an immune response to the antigen; and, (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, or identifying in the mouse of B a nucleic acid sequence encoding a heavy and/or light chain variable domain that binds the antigen, wherein the antibody comprises a light chain derived from a hVA, a hJA and a mouse CA gene.

[0121] The λ light chain constant gene is an intact mouse Cλ gene. In a specific aspect, the intact mouse Cλ gene is selected from CA1, CA2 and CA3. In a more specific aspect, the mouse Cλ gene is CA2.

[0122] Also disclosed is, a method for making a rearranged antibody gene in a genetically modified mouse is, comprising: (a) exposing a genetically modified mouse to an antigen, wherein the genetic modification comprises a hVA and a hJA at an endogenous light chain locus, wherein the endogenous light chain locus comprises an intact mouse Cλ gene; and, (b) identifying a rearranged immunoglobulin gene in said mouse, wherein the rearranged immunoglobulin gene comprises a λ light chain variable region gene segment and a CA gene or functional fragment thereof.

[0123] In one aspect, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region from the mouse, wherein the heavy and/or light chain variable region is from an antibody that comprises a human VA and a mouse CA.

[0124] [Deleted] [0125] [Deleted] [0126] [Deleted] [0127] [Deleted] [0128] In one aspect, a method for making a rearranged antibody gene in a genetically modified mouse is disclosed, comprising: (a) exposing a genetically modified mouse to an antigen, wherein the genetic modification comprises a hVA and a hJA at a mouse A light chain locus, wherein the A light chain locus comprises an intact mouse CA gene; and, (b) identifying a rearranged immunoglobulin gene in said mouse, wherein the rearranged immunoglobulin gene comprises a A light chain variable region gene segment and a CA gene.

[0129] The A light chain constant gene is an intact mouse CA gene.

[0130] In one aspect, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region from the mouse, wherein the heavy and/or light chain variable region is from an antibody that comprises a human VA and a mouse CA.

[0131] Also disclosed is a method for making an antibody, comprising exposing a mouse as described herein to an antigen, allowing the mouse to mount an immune response that comprises making an antibody that specifically binds the antigen, identifying a rearranged nucleic acid sequence in the mouse that encodes heavy chain and a rearranged nucleic acid sequence in the mouse that encodes a cognate light chain variable domain sequence of an antibody, wherein the antibody specifically binds the antigen, and employing the nucleic acid sequences of the heavy and light chain variable domains fused to human constant domains to make a desired antibody, wherein the desired antibody comprises a light chain that comprises a VA domain fused to a Cl domain. The VA domain is human and the Cl domain is a mouse CA domain.

[0132] [Deleted] [0133] In one aspect, a method for making an antibody is disclosed, comprising exposing a mouse as described herein to an antigen, allowing the mouse to mount an immune response that comprises making an antibody that specifically binds the antigen, identifying a rearranged nucleic acid sequence in the mouse that encodes a heavy chain variable domain and a rearranged nucleic acid sequence that encodes a cognate light chain variable domain sequence of an antibody, wherein the antibody specifically binds the antigen, and employing the nucleic acid sequences fused to nucleic acid sequences that encode a human heavy chain constant domain and a human light chain constant domain to make an antibody derived from human sequences, wherein the antibody that specifically binds the antigen comprises a light chain that comprises a human VA domain fused to a mouse CA region.

[0134] In one aspect, the mouse CA region is selected from CA1, CA2 and CA3. In a specific aspect, the mouse CA region is CA2.

[0135] In one aspect, a method for making a rearranged antibody light chain variable region gene sequence is disclosed, comprising (a) exposing a mouse as described herein to an antigen; (b) allowing the mouse to mount an immune response; (c) identifying a cell in the mouse that comprises a nucleic acid sequence that encodes a rearranged human VA domain sequence fused with a mouse CA domain, wherein the cell also encodes a cognate heavy chain comprising a human Vh domain and a mouse Ch domain, and wherein the cell expresses an antibody that binds the antigen; (d) cloning from the cell a nucleic acid sequence encoding the human VA domain and a nucleic acid sequence encoding the cognate human Vh domain; and, (e) using the cloned nucleic acid sequence encoding the human VA domain and the cloned nucleic acid sequence encoding the cognate human Vh domain to make a fully human antibody.

[0136] [Deleted] [0137] In one aspect, a method for making a rearranged antibody light chain variable region gene sequence is disclosed, comprising (a) exposing a mouse as described herein to an antigen; (b) allowing the mouse to mount an immune response to the antigen; (c) identifying a cell in the mouse that comprises DNAthat encodes a rearranged human VA domain sequence fused with a mouse CA domain, wherein the cell also encodes a cognate heavy chain comprising a human Vh domain and a mouse Ch domain, and wherein the cell expresses an antibody that binds the antigen; (d) cloning from the cell a nucleic acid sequence encoding the rearranged human VA domain and a nucleic acid sequence encoding the cognate human Vh domain; and, (e) using the cloned nucleic acid sequence encoding the human VA domain and the cloned nucleic acid sequence encoding the cognate human Vh domain to make a fully human antibody. In one aspect, the mouse CA domain is mouse CA2.

[0138] Also disclosed is a genetically modified mouse that expresses a human λ-derived light chain fused to an endogenous light chain constant region (CA), wherein the mouse, upon immunization with antigen, makes an antibody comprising a human VA domain fused to a mouse CA domain. In a specific aspect, the CA domain is CA2.

[0139] Also disclosed in a genetically modified mouse comprising a modified endogenous A light chain locus as described herein, that expresses a plurality of immunoglobulin A light chains associated with a plurality of immunoglobulin heavy chains. In one aspect, the heavy chain comprises a human sequence. In various aspects, the human sequence is selected from a variable sequence, a Ch1, a hinge, a Ch2, a Ch3, and a combination thereof. In one aspect, the plurality of immunoglobulin A light chains comprises a human sequence. In various aspects, the human sequence is selected from a variable sequence, a constant sequence, and a combination thereof. In one aspect, the mouse comprises a disabled endogenous immunoglobulin locus and expresses the heavy chain from a transgene or extrachromosomal episome. In one aspect, the mouse comprises a replacement at an endogenous mouse locus of some or all endogenous mouse heavy chain gene segments (/.e., V, D, J), and/or some or all endogenous mouse heavy chain constant sequences (e.g., Ch1, hinge, Ch2, Ch3, or a combination thereof), and/or some or all endogenous mouse light chain sequences {e.g., V, J, constant, or a combination thereof), with one or more human immunoglobulin sequences.

[0140] Also disclosed is a mouse suitable for making antibodies that have a human λ-derived light chain wherein all or substantially all antibodies made in the mouse are expressed with a human λ-derived light chain. In one aspect, the human λ-derived light chain is expressed from an endogenous light chain locus.

[0141] Also disclosed is a method for making a λ-derived light chain for a human antibody, comprising obtaining from a mouse as described herein a light chain sequence and a heavy chain sequence, and employing the light chain sequence and the heavy chain sequence in making a human antibody.

[0142] Also disclosed is a method for making an antigen-binding protein, comprising exposing a mouse as described herein to an antigen; allowing the mouse to mount an immune response; and obtaining from the mouse an antigen-binding protein that binds the antigen, or obtaining from the mouse a sequence to be employed in making an antigen-binding protein that binds the antigen.

[0143] Also disclosed is a cell derived from a mouse as described herein. In one aspect, the cell is selected from an embryonic stem cell, a pluripotent cell, an induced pluripotent cell, a B cell, and a hybridoma.

[0144] Also disclosed is a cell that comprises a genetic modification as described herein. In one aspect, the cell is a mouse cell. In one aspect, the cell is selected from a hybridoma and a quadroma. In one aspect, the cell expresses an immunoglobulin light chain that comprises a human λ variable sequence fused with a mouse λ constant sequence.

[0145] Also disclosed is a tissue derived from a mouse as described herein.

[0146] Also disclosed is, use of a mouse or a cell as described herein to make an antigen-binding protein. In one aspect, the antigen-binding protein is a human protein. In one aspect, the human protein is a human antibody.

[0147] Also disclosed is an antigen-binding protein made by a mouse, cell, tissue, or method as described herein. In one aspect, the antigen-binding protein is a human protein. In one aspect, the human protein is a human antibody.

[0148] [Deleted]

BRIEF DESCRIPTION OF THE FIGURES

[0149] FIG. 1 shows a detailed illustration, not to scale, of the human λ light chain locus including the clusters of \/λ gene segments (A, B and C) and the ύλ and CK region pairs (J-C pairs) FIG. 2 shows a general illustration, not to scale, of a targeting strategy used to inactivate the endogenous mouse λ light chain locus. FIG. 3 shows a general illustration, not to scale, of a targeting strategy used to inactivate the endogenous mouse κ light chain locus. FIG. 4A shows a general illustration, not to scale of an initial targeting vector for targeting the endogenous mouse λ light chain locus with human λ light chain sequences including 12 hN/λ gene segments and h^1 gene segment (12/1-λ Targeting Vector). FIG. 4B shows a general illustration, not to scale, of four initial targeting vectors for targeting the endogenous mouse κ light chain locus with human λ light chain sequences including 12 hM gene segments and h^1 gene segment (12/1-k Targeting Vector), 12 ΙΆ/λ gene segments and Κϋλ1, 2, 3 and 7 gene segments (12/4-k Targeting Vector), 12 ΙΆ/λ gene segments, a human Vk-Jk genomic sequence and h^1 gene segment (12(k)1-k Targeting Vector) and 12 ΙΆ/λ gene segments, a human Vk-Jk genomic sequence and Ιπύλΐ, 2, 3 and 7 gene segments (12(k)4-k Targeting Vector). FIG. 5A shows a general illustration, not to scale, of a targeting strategy for progressive insertion of 40 ΙΆ/λ gene segments and a single Ιπύλ gene segment into the mouse λ light chain locus. FIG. 5B shows a general illustration, not to scale, of a targeting strategy for progressive insertion of 40 Ιινλ gene segments and a single Ιπύλ gene segment into the mouse κ locus. FIG. 6 show a general illustration, not to scale, of the targeting and molecular engineering steps employed to make unique human λ-κ hybrid targeting vectors for construction of a hybrid light chain locus containing a human κ intergenic sequence, multiple hJA gene segments or both. FIG. 7 A shows a general illustration, not to scale, of the locus structure for a modified mouse A light chain locus containing 40 hVA gene segments and a single hJA gene segment operably linked to the endogenous CA2 gene. FIG. 7B shows a general illustration, not to scale, of the locus structure for four independent, modified mouse κ light chain loci containing 40 hVA gene segments and either one or four hJA gene segments with or without a contiguous human Vk-Jk genomic sequence operably linked to the endogenous Ck gene. FIG. 8A shows contour plots of lgA+ and lgK+ splenocytes gated on CD19+ from a wild type mouse (WT), a mouse homozygous for 12 hVA and four hJA gene segments including a human Vk-Jk genomic sequence (12IiVA-Vk4Jk) and a mouse homozygous for 40 hVA and one hJA gene segment (40hVA-1hJA). FIG. 8B shows the total number of CD19+ B cells in harvested spleens from wild type (WT), mice homozygous for 12 hVA and four hJA gene segments including a human Vk-Jk genomic sequence (12hVA-VKjK-4hJA) and mice homozygous for 40 hVA and one hJA gene segment (40hVA-1hJA). FIG. 9A, in the top panel, shows contour plots of splenocytes gated on singlets and stained for B and T cells (CD19+ and CD3+, respectively) from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four Jλ gene segments including a human

Vk-Jk genomic sequence (40hVA-VKjK-4hJA). The bottom panel shows contour plots of splenocytes gated on CD19+ and stained for lgA+ and lgK+ expression from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 9B shows the total number of CD19+, CD19+lgK+ and CD19+lgA+ B cells in harvested spleens from wild type mice (WT) and mice homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 9C shows contour plots of splenocytes gated on CD19+ and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). Mature (72 for WT, 51 for 40hVA-VKjK-4hJA) and transitional (13 for WT, 22 for 40hVA-VKjK-4hJA) B cells are noted on each of the contour plots. FIG. 9D shows the total number of CD19+ B cells, transitional B cells (CD19+lgM'1'lgD'°) and mature B cells (CD19+lgM'°lgD^) in harvested spleens from wild type mice (WT) and mice homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 10A, in the top panel, shows contour plots of bone marrow stained for B and T cells (CD19+ and CD3+, respectively) from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). The bottom panel shows contour plots of bone marrow gated on CD19+ and stained for ckit+ and CD43+ from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four J λ gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). Pro and Pre B cells are noted on the contour plots of the bottom panel. FIG. 10B shows the number of Pro (CD19+CD43+ckit+) and Pre (CD19+CD43‘ ckit') B cells in bone marrow harvested from the femurs of wild type mice (WT) and mice homozygous for 40 hVA and four JA gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 10C shows contour plots of bone marrow gated on singlets stained for immunoglobulin M (IgM) and B220 from a wild type mouse (WT) and a mouse homozygous for 40 hVA and four JA gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). Immature, mature and pro/pre B cells are noted on each of the contour plots. FIG. 10D shows the total number of immature (B220intlgM+) and mature (B220hilgM+) B cells in bone marrow isolated from the femurs of wild type mice (WT) and mice homozygous for 40 hVA and four JA gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 10E shows contour plots of bone marrow gated on immature (B220intlgM+) and mature (B220hilgM+) B cells stained for IgA and IgK expression isolated from the femurs of a wild type mouse (WT) and a mouse homozygous for 40 hVA and four JA gene segments including a human Vk-Jk genomic sequence (40hVA-VKjK-4hJA). FIG. 11 shows a nucleotide sequence alignment of the VA-JA-Ck junction of eighteen independent RT-PCR clones amplified from splenocyte RNAof mice bearing human A light chain gene sequences at an endogenous mouse κ light chain locus. A6 = SEQ ID NO:57; B6 = SEQ ID NO:58; F6 = SEQ ID NO:59; B7 = SEQ ID NO:60; E7 = SEQ ID NO:61; F7 = SEQ ID NO:62; C8 = SEQ ID NO:63; E12 = SEQ ID NO:64; 1-4 = SEQ ID NO:65; 1-20 = SEQ ID NO:66; 3B43 = SEQ ID NO:67; 5-8 = SEQ ID NO:68; 5-19 = SEQ ID NO:69; 1010 = SEQ ID NO:70; 11A1 = SEQ ID NO:71; 7A8 = SEQ ID NO:72; 3A3 = SEQ ID NO:73; 2-7 = SEQ ID NO:74. Lower case bases indicate non-germline bases resulting from either mutation and/or N addition during recombination. Consensus amino acids within the Framework 4 region (FWR4) encoded by the nucleotide sequence of hJA1 and mouse Ck are noted at the bottom of the sequence alignment. FIG. 12 shows a nucleotide sequence alignment of the VA-JA-Ck junction of twelve independent RT-PCR clones amplified from splenocyte RNAof mice bearing human A light chain gene sequences including a contiguous human Vk-Jk genomic sequence at an endogenous mouse κ light chain locus. 5-2 = SEQ ID NO:87; 2-5 = SEQ ID NO:88; 1-3 = SEQ ID NO:89; 4B-1 = SEQ ID NO:90; 3B-5 = SEQ ID NO:91; 7A-1 = SEQ ID NO:92; 5-1 = SEQ ID NO:93; 4A-1 = SEQ ID NO:94; 11A-1 = SEQ ID NO:95; 5-7 = SEQ ID NO:96; 5-4 = SEQ ID NO:97; 2-3 = SEQ ID NO:98. Lower case bases indicate non-germline bases resulting from either mutation and/or N addition during recombination. Consensus amino acids within the Framework 4 region (FWR4) encoded by the nucleotide sequence of each human JA and mouse Ck are noted at the bottom of the sequence alignment. FIG. 13 shows a nucleotide sequence alignment of the VA-JA-Ck junction of three independent RT-PCR clones amplified from splenocyte RNAof mice bearing human A light chain gene sequences at an endogenous mouse A light chain locus. 2D1 = SEQ ID NO:101; 2D9 = SEQ ID NO:102; 3E15 = SEQ ID NO:103. Lower case bases indicate non-germline bases resulting from either mutation and/or N addition during recombination. Consensus amino acids within the Framework 4 region (FWR4) encoded by the nucleotide sequence of hJA1 and mouse CA2 are noted at the bottom of the sequence alignment.

DETAILED DESCRIPTION

[0150] The invention in its broadest sense is as defined in the independent claims.

[0151] The term "contiguous" includes reference to occurrence on the same nucleic acid molecule, e.g., two nucleic acid sequences are "contiguous" if they occur on the same nucleic molecule but are interrupted by another nucleic acid sequence. For example, a rearranged V(D)J sequence is "contiguous" with a constant region gene sequence, although the final codon of the V(D)J sequence is not followed immediately by the first codon of the constant region sequence. In another example, two V gene segment sequences are "contiguous" if they occur on the same genomic fragment, although they may be separated by sequence that does not encode a codon of the V region, e.g., they may be separated by a regulatory sequence, e.g., a promoter or other noncoding sequence. In one embodiment, a contiguous sequence includes a genomic fragment that contains genomic sequences arranged as found in a wild-type genome.

[0152] The phrase "derived from" when used concerning a variable region "derived from" a cited gene or gene segment includes the ability to trace the sequence back to a particular unrearranged gene segment or gene segments that were rearranged to form a gene that expresses the variable domain (accounting for, where applicable, splice differences and somatic mutations).

[0153] The phrase "functional" when used concerning a variable region gene segment or joining gene segment refers to usage in an expressed antibody repertoire; e.g., in humans VA gene segments 3-1, 4-3, 2-8, etc. are functional, whereas VA gene segments 3-2, 3-4, 2-5, etc. are nonfunctional.

[0154] A "heavy chain locus" includes a location on a chromosome, e.g., a mouse chromosome, wherein in a wild-type mouse heavy chain variable (Vh), heavy chain diversity (Dh), heavy chain joining (Jh), and heavy chain constant (Ch) region DNA sequences are found.

[0155] A "k locus" includes a location on a chromosome, e.g., a mouse chromosome, wherein in a wild-type mouse Kvariable (Vk), k joining (Jk), and κ constant (Ck) region DNA sequences are found.

[0156] A "A locus" includes a location on a chromosome, e.g., a mouse chromosome, wherein in a wild-type mouse A variable (VA), A joining (JA), and A constant (CK) region DNA sequences are found.

[0157] The term "unrearranged" includes the state of an immunoglobulin locus wherein V gene segments and J gene segments (for heavy chains, D gene segments as well) are maintained separately but are capable of being joined to form a rearranged V(D)J gene that comprises a single V,(D),J of the V(D)J repertoire.

Mice Expressing Human λ Variable Domains [0158] Mice that express antibodies that are fully human, or partly human and partly mouse, have previously been reported. VELOCIMMUNE® genetically engineered mice comprise a replacement of unrearranged V(D)J gene segments at endogenous mouse loci with human V(D)J gene segments. VELOCIMMUNE® mice express chimeric antibodies having human variable domains and mouse constant domains (see, e.g., US Pat. No. 7,605,237). Most other reports concern mice that express fully human antibodies from fully human transgenes in mice that have disabled endogenous immunoglobulin loci.

[0159] Antibody light chains are encoded by one of two separate loci: kappa (k) and lambda (A). Mouse antibody light chains are primarily of the κ type. Mice that make mouse antibodies, and modified mice that make fully human or chimeric human-mouse antibodies, display a bias in light chain usage. Humans also exhibit light chain bias, but not so pronounced as in mice; the ratio of κ light chains to A light chains in mice is about 95:5, whereas in humans the ratio is about 60:40. The more pronounced bias in mice is not thought to severely affect antibody diversity, because in mice the A variable locus is not so diverse in the first instance. This is not so in humans. The human A light chain locus is richly diverse.

[0160] The human A light chain locus extends over 1,000 kb and contains over 80 genes that encode variable (V) or joining (J) segments (FIG. 1). Within the human A light chain locus, over half of all observed VA domains are encoded by the gene segments 1-40, 1-44, 2-8, 2-14, and 3-21. Overall, about 30 or so of the human VA gene segments are believed to be functional. There are seven JA gene segments, only four of which are regarded as generally functional JA gene segments JA1, JA2, JA3, and JA7.

[0161] The A light chain locus in humans is similar in structure to the κ locus of both mice and humans in that the human A light chain locus has several variable region gene segments that are capable of recombining to form a functional light chain protein. The human A light chain locus contains approximately 70 V gene segments and 7 JA-CA gene segment pairs. Only four of these JA-CA gene segment pairs appear to be functional. In some alleles, a fifth JA-CA gene segment pair is reportedly a pseudo gene (CA6). The 70 VA gene segments appear to contain 38 functional gene segments. The 70 VA sequences are arranged in three clusters, all of which contain different members of distinct V gene family groups (clusters A, B and C; FIG. 1). This is a potentially rich source of relatively untapped diversity for generating antibodies with human V regions in non-human animals.

[0162] In stark contrast, the mouse A light chain locus contains only two or three (depending on the strain) mouse VA region gene segments (FIG. 2). At least for this reason, the severe κ bias in mice is not thought to be particularly detrimental to total antibody diversity.

[0163] According published maps of the mouse A light chain locus, the locus consists essentially of two clusters of gene segments within a span of approximately 200 kb (FIG.2). The two clusters contain two sets of V, J, and C genes that are capable of independent rearrangement: VA2-JA2-CA2-JA4-CA4 and VA1-JA3-CA3-JA1-CA1. Although VA2 has been found to recombine with all JA gene segments, VA1 appears to exclusively recombine with CA1. CA4 is believed to be a pseudo gene with defective splice sites.

[0164] The mouse κ light chain locus is strikingly different. The structure and number of gene segments that participate in the recombination events leading to a functional light chain protein from the mouse κ locus is much more complex (FIG. 3). Thus, mouse A light chains do not greatly contribute to the diversity of an antibody population in a typical mouse.

[0165] Exploiting the rich diversity of the human A light chain locus in mice would likely result in, among other things, a source for a more complete human repertoire of light chain V domains. Previous attempts to tap this diversity used human transgenes containing chunks of the human A light chain locus randomly incorporated into the mouse genome (see, e.g., US 6,998,514 and US 7,435,871). Mice containing these randomly integrated transgenes reportedly express fully human A light chains, however, in some cases, one or both endogenous light chain loci remain intact. This situation is not desirable as the human A light chain sequences contend with the mouse light chain (κ or A) in the expressed antibody repertoire of the mouse.

[0166] In contrast, the inventors describe genetically modified mice that are capable of expressing one or more A light chain nucleic acid sequences directly from an endogenous lambda mouse light chain locus. Genetically modified mice capable of expressing human A light chain sequences from an endogenous A locus may be further bred to mice that comprise a human heavy chain locus and thus be used to express antibodies comprising V regions (heavy and A light) that are fully human. The V regions express with mouse constant regions. In various aspects, no endogenous mouse immunoglobulin gene segments are present and the V regions express with human constant regions. These antibodies would prove useful in numerous applications, both diagnostic as well as therapeutic.

[0167] Many advantages can be realized for various aspects of expressing binding proteins derived from human VA and Jλ gene segments in mice. Advantages can be realized by placing human λ sequences at the endogenous λ light chain locus. Antibodies made from such mice can have light chains that comprise human VA domains fused to a mouse mouse CA region. The mice will also express human VA domains that are suitable for identification and cloning for use with human Cl regions, specifically Ck and/or CA regions. Because B cell development in such mice is otherwise normal, it is possible to generate compatible VA domains (including somatically mutated VA domains) in the context of either CA or Ck regions.

[0168] Genetically modified mice are described that comprise an unrearranged human VA gene segment and an unrearranged human JA at an endogenous immunoglobulin A light chain locus. Mice that express antibodies that comprise a light chain having a human VA domain fused to a CA region are described.

[0169] [Deleted] [0170] [Deleted] [0171] [Deleted] [0172] [Deleted] [0173] [Deleted] [0174] [Deleted]

Approaches to Engineering Mice to Express Human VA Domains [0175] Various approaches to making genetically modified mice that make antibodies that contain a light chain that has a human VA domain fused to an endogenous CA region are described. Genetic modifications are described that, in various aspects, comprise a deletion of one or both endogenous light chain loci. For example, to eliminate mouse A light chains from the endogenous antibody repertoire a deletion of a first VA-JA-CA gene cluster and replacement, in whole or in part, of the VA-JA gene segments of a second gene cluster with human VA-JA gene segments can be made as recited in the claims. Genetically modified mouse embryos, cells, and targeting constructs for making the mice, mouse embryos, and cells are also disclosed.

[0176] The deletion of one endogenous VA-JA-CA gene cluster and replacement of the VA-JA gene segments of another endogenous VA-JA-CA gene cluster employs a relatively minimal disruption in natural antibody constant region association and function in the animal, in various aspects, because endogenous CA genes are left intact and therefore retain normal functionality and capability to associate with the constant region of an endogenous heavy chain. Thus, in such aspects the modification does not affect other endogenous heavy chain constant regions dependent upon functional light chain constant regions for assembly of a functional antibody molecule containing two heavy chains and two light chains. Further, in various aspects the modification does not affect the assembly of a functional membrane-bound antibody molecule involving an endogenous heavy chain and a light chain, e.g., a hVA domain linked to a mouse CA region. Because at least one functional CA gene is retained at the endogenous locus, animals containing a replacement of the VA-JA gene segments of an endogenous VA-JA-CA gene cluster with human VA-JA gene segments should be able to make normal A light chains that are capable of binding antigen during an immune response through the human VA-JA gene segments present in the expressed antibody repertoire of the animal.

[0177] A schematic illustration (not to scale) of a deleted endogenous mouse VA-JA-CA gene cluster is provided in FIG. 2. As illustrated, the mouse A light chain locus is organized into two gene clusters, both of which contain function gene segments capable of recombining to form a function mouse A light chain. The endogenous mouse VA1-JA3-CA3-JA1-CA1 gene cluster is deleted by a targeting construct (Targeting Vector 1) with a neomycin cassette flanked by recombination sites. The other endogenous gene cluster (VA2-VA3-JA2-CA2-JA4-CA4) is deleted in part by a targeting construct (Targeting Vector 2) with a hygromycin-thymidine kinase cassette flanked by recombination sites. In this second targeting event, the CA2-JA4-CA4 endogenous gene segments are retained. The second targeting construct (Targeting Vector 2) is constructed using recombination sites that are different than those in the first targeting construct (Targeting Vector 1) thereby allowing for the selective deletion of the selection cassette after a successful targeting has been achieved. The resulting double-targeted locus is functionally silenced in that no endogenous A light chain can be produced. This modified locus can be used for the insertion of human VA and Jλ gene segments to create an endogenous mouse λ locus comprising human VA and JA gene segments, whereby, upon recombination at the modified locus, the animal produces A light chains comprising rearranged human VA and JA gene segments linked to an endogenous mouse CA gene segment.

[0178] Genetically modifying a mouse to render endogenous A gene segments nonfunctional, in various aspects, results in a mouse that exhibits exclusively κ light chains in its antibody repertoire, making the mouse useful for evaluating the role of A light chains in the immune response, and useful for making an antibody repertoire comprising Vk domains but not VA domains.

[0179] A genetically modified mouse that expresses a hVA linked to a mouse CA gene having been recombined at the endogenous mouse A light chain locus can be made by any method known in the art. A schematic illustration (not to scale) of the replacement of the endogenous mouse VA2-VA3-JA2 gene segments with human VA and JA gene segments is provided in FIG. 4A. As illustrated, an endogenous mouse A light chain locus that had been rendered nonfunctional is replaced by a targeting construct (12/1-A Targeting Vector) that includes a neomycin cassette flanked by recombination sites. The VA2-VA3-JA2 gene segments are replaced with a genomic fragment containing human A sequence that includes 12 hVA gene segments and a single hJA gene segment.

[0180] Thus, this first approach positions one or more hVA gene segments at the endogenous A light chain locus contiguous with a single hJA gene segment (FIG. 4A).

[0181] Further modifications to the modified endogenous A light chain locus can be achieved with using similar techniques to insert more hVA gene segments. For example, schematic illustrations of two additional targeting constructs (+16-A and +12-A Targeting Vectors) used for progressive insertion of addition human hVA gene segments are provided in FIG. 5A. As illustrated, additional genomic fragments containing specific human hVA gene segments are inserted into the modified endogenous A light chain locus in successive steps using homology provided by the previous insertion of human A light chain sequences. Upon recombination with each targeting construct illustrated, in sequential fashion, 28 additional hVA gene segments are inserted into the modified endogenous A light chain locus. This creates a chimeric locus that produces a A light chain protein that comprises human VA-JA gene segments linked to a mouse CA gene.

[0182] The above approaches to insert human A light chain gene segments at the mouse A locus, maintains the enhancers positioned downstream of the CA2-JA4-CA4 gene segments (designated Enh 2.4, Enh and Enh 3.1 FIG. 4Aand FIG. 5A). This approach results in a single modified allele at the endogenous mouse A light chain locus (FIG. 7A).

[0183] Also disclosed are compositions and methods for making a mouse that expresses a light chain comprising hVA and JA gene segments operably linked to a mouse CA gene segment, including compositions and method for making a mouse that expresses such genes from an endogenous mouse A light chain locus. The methods include selectively rendering one endogenous mouse VA-JA-CA gene cluster nonfunctional (e.g., by a targeted deletion), and employing a hVA and JA gene segments at the endogenous mouse A light chain locus to express a hVA domain in a mouse.

[0184] Also disclosed is a second approach where human A light chain gene segments may be positioned at the endogenous κ light chain locus. The genetic modification, in various aspects, comprises a deletion of the endogenous κ light chain locus. For example, to eliminate mouse κ light chains from the endogenous antibody repertoire a deletion of the mouse Vk and Jk gene segments can be made. Genetically modified mouse embryos, cells, and targeting constructs for making the mice, mouse embryos, and cells are also disclosed.

[0185] For the reasons stated above, the deletion of the mouse Vk and Jk gene segments employs a relatively minimal disruption. A schematic illustration (not to scale) of deleted mouse Vk and Jk gene segments is provided in FIG. 3. The endogenous mouse Vk and Jk gene segments are deleted via recombinase-mediated deletion of mouse sequences position between two precisely positioned targeting vectors each employing site-specific recombination sites. A first targeting vector (Jk Targeting Vector) is employed in a first targeting event to delete the mouse Jk gene segments. A second targeting vector (Vk Targeting Vector) is employed in a second, sequential targeting event to delete a sequence located 5' of the most distal mouse Vk gene segment. Both targeting vectors contain site-specific recombination sites thereby allowing for the selective deletion of both selection cassettes and all intervening mouse κ light chain sequences after a successful targeting has been achieved. The resulting deleted locus is functionally silenced in that no endogenous κ light chain can be produced. This modified locus can be used for the insertion of hVA and JA gene segments to create an endogenous mouse κ locus comprising hVA and JA gene segments, whereby, upon recombination at the modified locus, the animal produces A light chains comprising rearranged hVA and JA gene segments operably linked to an endogenous mouse Ck gene segment. Various targeting vectors comprising human A light chain sequences can be used in conjunction with this deleted mouse κ locus to create a hybrid light chain locus containing human λ gene segments operably linked with a mouse Ck region.

[0186] Thus, a second approach positions one or more human N/λ gene segments are positioned at the mouse κ light chain locus contiguous with a single human Jλ gene segment (12/1-k Targeting Vector, FIG. 4B).

[0187] Modifications to this approach can be made to add gene segments and/or regulatory sequences to optimize the usage of the human λ light chain sequences from the mouse κ locus within the mouse antibody repertoire.

[0188] Also disclosed is a third approach where, one or more hVA gene segments are positioned at the mouse κ light chain locus contiguous with four hJA gene sequences (12/4-k Targeting Vector FIG. 4B).

[0189] In a third approach, one or more hVA gene segments are positioned at the mouse κ light chain locus contiguous with a human κ intergenic sequence and a single hJA gene sequence (12(k)1-k Targeting Vector, FIG. 4B).

[0190] Also disclosed is a fourth approach, one or more hVA gene segments are positioned at the mouse κ light chain locus contiguous with a human κ intergenic sequence four hJA gene sequences (12(k)4-k Targeting Vector FIG. 4B).

[0191] All of the above approaches to insert human λ light chain gene segments at the mouse κ locus, maintain the κ intronic enhancer element upstream of the Ck gene (designated Εκί, FIG. 4B and FIG. 5B) and the 3' κ enhancer downstream of the Ck gene (designated Ek3', FIG. 4B and FIG. 5B). The approaches result in four separate modified alleles at the endogenous mouse κ light chain locus (FIG. 7B).

[0192] [Deleted]

Lambda Domain Antibodies from Genetically Modified Mice [0193] Mice comprising human λ sequences at the mouse λ light chain locus will express a light chain that comprises a hVA region fused to a mouse Cλ region. These are advantageously bred to mice that (a) comprise a functionally silenced light chain locus (e.g., a knockout of the endogenous mouse κ or λ light chain locus); (b) comprise an endogenous mouse λ light chain locus that comprises hVA and hJA gene segments operably linked to an endogenous mouse Cλ gene as recited in the claims; (c) comprise an endogenous mouse κ light chain locus that comprises hVK and hJK gene segments operably linked to an endogenous mouse Ck gene; and, (d) a mouse in which one κ allele comprises hVKS and hJKs; the other κ allele comprising hVAs and hJAs; one λ allele comprising hVAs and hJAs and one λ allele silenced or knocked out, or both λ alleles comprising hVAs and hJAs*; and, two heavy chain alleles that each comprise hVns, hDns, and hJns.

[0194] The antibodies that comprise the hVA domains expressed in the context of either CK are used to make fully human antibodies by cloning the nucleic acids encoding the hVA domains into expression constructs that bear genes encoding human CK. Resulting expression constructs are transfected into suitable host cells for expressing antibodies that display a fully hVA domain fused to hCA.

EXAMPLES

[0195] The following examples are provided so as to describe how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, temperature is indicated in Celsius, and pressure is at or near atmospheric.

Example I

Deletion of the Mouse Immunoglobulin Light Chain Loci [0196] Various targeting constructs were made using VELOCIGENE® technology (see, e.g. , US Pat. No. 6,586,251 and

Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659) to modify mouse genomic Bacterial Artificial Chromosome (BAC) libraries to inactivate the mouse k and A light chain loci.

[0197] Deletion of the mouse λ light chain locus. DNAfrom mouse BAC clone RP23-135k15 (Invitrogen) was modified by homologous recombination to inactivate the endogenous mouse A light chain locus through targeted deletion of the VA-JA-CA gene clusters (FIG. 2).

[0198] Briefly, the entire proximal cluster comprising VA1-JA3-CA3-JA1-CA1 gene segments was deleted in a single targeting event using a targeting vector comprising a neomycin cassette flanked by /oxP sites with a 5' mouse homology arm containing sequence 5' of the VA1 gene segment and a 3' mouse homology arm containing sequence 3' of the CA1 gene segment (FIG. 2, Targeting Vector 1).

[0199] A second targeting construct was prepared to precisely delete the distal endogenous mouse A gene cluster containing VA2-JA2-CA2-JA4-CA4 except that the targeting construct contained a 5' mouse homology arm that contained sequence 5' of the VA2 gene segment and a 3' mouse homology arm that contained sequence 5' to the endogenous CA2 gene segment (FIG. 2, Targeting Vector 2). Thus, the second targeting construct precisely deleted VA2-JA2, while leaving CA2-JA4-CA4 intact at the endogenous mouse A locus. ES cells containing an inactivated endogenous A locus (as described above) were confirmed by karyotyping and screening methods (e.g., TAQMAN®) known in the art. DNAwas then isolated from the modified ES cells and subjected to treatment with CRE recombinase thereby mediating the deletion of the proximal targeting cassette containing the neomycin marker gene, leaving only a single loxP site at the deletion point (FIG. 2, bottom).

[0200] Deletion of the mouse κ light chain locus. Several targeting constructs were made using similar methods described above to modify DNAfrom mouse BAC clones RP23-302g12 and RP23-254m04 (Invitrogen) by homologous recombination to inactivate the mouse κ light chain locus in a two-step process (FIG. 3).

[0201] Briefly, the Jk gene segments (1-5) of the endogenous mouse κ light chain locus were deleted in a single targeting event using a targeting vector comprising a hygromycin-thymidine kinase (hyg-TK) cassette containing a single /oxP site 3' to the hyg-TK cassette (FIG. 3, Jk Targeting Vector). The homology arms used to make this targeting vector contained mouse genomic sequence 5' and 3' of the endogenous mouse Jk gene segments. In a second targeting event, a second targeting vector was prepared to delete a portion of mouse genomic sequence upstream (5') to the most distal endogenous mouse Vk gene segment (FIG. 3, Vk Targeting Vector). This targeting vector contained an inverted /ox511 site, a /oxP site and a neomycin cassette. The homology arms used to make this targeting vector contained mouse genomic sequence upstream of the most distal mouse Vk gene segment. The targeting vectors were used in a sequential fashion (/.e., Jk then Vk) to target DNAin ES cells. ES bearing a double-targeted chromosome (/.e., a single endogenous mouse κ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., Taqman™) known in the art. DNAwas then isolated from the modified ES cells and subjected to treatment with Cre recombinase thereby mediating the deletion of endogenous mouse Vk gene segments and both selection cassettes, while leaving two juxtaposed /ox sites in opposite orientation relative to one another (FIG. 3, bottom; SEQ ID NO:1).

[0202] Thus, two modified endogenous light chain loci (κ and A) containing intact enhancer and constant regions were created for progressively inserting unrearranged human A germline gene segments in a precise manner using targeting vectors described below.

Example II

Replacement of Mouse Light Chain Loci with a Human λ Light Chain Mini-Locus [0203] Multiple targeting vectors were engineered for progressive insertion of human A gene segments into the endogenous mouse κ and A light chain loci using similar methods as described above. Multiple independent initial modifications were made to the endogenous light chain loci each producing a chimeric light chain locus containing hVA and JA gene segments operably linked to mouse light chain constant genes and enhancers.

[0204] A human λ mini-locus containing 12 human VA and one human JA gene segment. A series of initial targeting vectors were engineered to contain the first 12 consecutive human VA gene segments from cluster A and a hJA1 gene segment or four hJA gene segments using a human BAC clone named RP11-729g4 (Invitrogen). FIGs. 4Aand 4B show the targeting vectors that were constructed for making an initial insertion of human λ light chain gene segments at the mouse λ and κ light chain loci, respectively.

[0205] For a first set of initial targeting vectors, a 124,125 bp DNAfragment from the 729g4 BAC clone containing 12 hVA gene segments and a hJA1 gene segment was engineered to contain a Pl-Scel site 996 bp downstream (3') of the hJA1 gene segment for ligation of a 3' mouse homology arm. Two different sets of homology arms were used for ligation to this human fragment; one set of homology arms contained endogenous mouse λ sequences from the 135k15 BAC clone (FIG.4A) and another set contained endogenous κ sequence 5' and 3' of the mouse Vk and Jk gene segments from mouse BAC clones RP23-302g12 and RP23-254m04, respectively (FIG. 4B).

[0206] For the 12/1 -λ Targeting Vector (FIG. 4A), a Pl-Scel site was engineered at the 5' end of a 27,847 bp DNA fragment containing the mouse CA2-JA4-CA4 and enhancer 2.4 of the modified mouse λ locus described in Example 1. The -28 kb mouse fragment was used as a 3' homology arm by ligation to the -124 kb human λ fragment, which created a 3' junction containing, from 5' to 3', a hJA1 gene segment, 996 bp of human λ sequence 3' of the hJA1 gene segment, 1229 bp of mouse λ sequence 5' to the mouse CA2 gene, the mouse CA2 gene and the remaining portion of the -28 kb mouse fragment. Upstream (5') from the human VA3-12 gene segment was an additional 1456 bp of human λ sequence before the start of the 5' mouse homology arm, which contained 23,792 bp of mouse genomic DNA corresponding to sequence 5' of the endogenous mouse λ locus. Between the 5' homology arm and the beginning of the human λ sequence was a neomycin cassette flanked by Frt sites.

[0207] Thus, the 12/1-λ Targeting Vector included, from 5' to 3', a 5' homology arm containing -24 kb of mouse λ genomic sequence 5' of the endogenous λ locus, a 5' Frt site, a neomycin cassette, a 3' Frt site, -123 kb of human genomic λ sequence containing the first 12 consecutive hVA gene segments and a hJA1 gene segment, a Pl-Scel site, and a 3' homology arm containing -28 kb of mouse genomic sequence including the endogenous CA2-JA4-CA4 gene segments, the mouse enhancer 2.4 sequence and additional mouse genomic sequence downstream (3') of the enhancer 2.4 (FIG. 4A).

[0208] In a similar fashion, the 12/1 -κ Targeting Vector (FIG. 4B) employed the same -124 human λ fragment with the exception that mouse homology arms containing mouse κ sequence were used such that targeting to the endogenous κ locus could be achieved by homologous recombination. Thus, the 12/1 -κ Targeting Vector included, from 5' to 3', a 5' homology arm containing -23 kb of mouse genomic sequence 5' of the endogenous κ locus, an l-Ceul site, a 5' Frt site, a neomycin cassette, a 3' Frt site, -124 kb of human genomic λ sequence containing the first 12 consecutive hVA gene segments and a hJA1 gene segment, a Pl-Scel site, and a 3' homology arm containing -28 kb of mouse genomic sequence including the endogenous the mouse Ck gene, Εκί and Ek3' and additional mouse genomic sequence downstream (3') of Ek3' (FIG. 4B, 12/1 -κ Targeting Vector).

[0209] Pbmologous recombination with either of these two initial targeting vectors created a modified mouse light chain locus (κ or λ) containing 12 hVA gene segments and a hJA1 gene segment operably linked to the endogenous mouse light chain constant gene and enhancers (Ck or Ck2 and Εκί/Εκ3' or Enh 2.4/Enh 3.1) gene which, upon recombination, leads to the formation of a chimeric λ light chain.

[0210] A human λ mini-locus with 12 human VA and four human JA gene segments. In another approach to add diversity to a chimeric λ light chain locus, a third initial targeting vector was engineered to insert the first 12 consecutive human VA gene segments from cluster A and hJA1, 2, 3 and 7 gene segments into the mouse κ light chain locus (FIG. 4B, 12/4-k Targeting Vector). A DNA segment containing hJA1, JA2, JA3 and JA7 gene segments was made by de novo DNA synthesis (Integrated DNA Technologies) including each JA gene segment and human genomic sequence of -100 bp from both the immediate 5' and 3' regions of each JA gene segment. A Pl-Scel site was engineered into the 3' end of this -1 kb DNA fragment and ligated to a chloroamphenicol cassette. Plomology arms were PCR amplified from human A sequence at 5' and 3' positions relative to the hJA1 gene segment of the human BAC clone 729g4. Plomologous recombination with this intermediate targeting vector was performed on a modified 729g4 BAC clone that had been previously targeted upstream (5') of the human VA3-12 gene segment with a neomycin cassette flanked by Frt sites, which also contained an l-Ceul site 5' to the 5' Frt site. The double-targeted 729g4 BAC clone included from 5' to 3' an l-Ceul site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a -123 kb fragment containing the first 12 hVA gene segments, a -1 kb fragment containing human JA1, 2, 3 and 7 gene segments, a Pl-Scel site, and a chloroamphenicol cassette. This intermediate targeting vector was digested together with l-Ceul and Pl-Scel and subsequently ligated into the modified mouse BAC clone (described above) to create the third targeting vector.

[0211] This ligation resulted in a third targeting vector for insertion of human A sequences into the endogenous κ light chain locus, which included, from 5' to 3', a 5' mouse homology arm containing -23 kb of genomic sequence 5' of the endogenous mouse κ locus, an l-Ceul site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a -123 kb fragment containing the first 12 hVA gene segments, a -1 kb fragment containing hJA1,2, 3 and 7 gene segments, a Pl-Scel site and a 3' homology arm containing -28 kb of mouse genomic sequence including the endogenous the mouse Ck gene, Εκί and Ek3' and additional mouse genomic sequence downstream (3') of Ek3' (FIG. 4B, 12/4-k Targeting Vector). Homologous recombination with this third targeting vector created a modified mouse κ light chain locus containing 12 hVA gene segments and four hJA gene segments operably linked to the endogenous mouse Ck gene which, upon recombination, leads to the formation of a chimeric human λ/mouse κ light chain.

[0212] A human λ mini-locus with an integrated human κ light chain sequence. In a similar fashion, two additional targeting vectors similar to those engineered to make an initial insertion of human λ gene segments into the endogenous κ light chain locus (FIG. 4B, 12/1 -κ and 12/4-k Targeting Vectors) were engineered to progressively insert human λ light chain gene segments using uniquely constructed targeting vectors containing contiguous human λ and κ genomic sequences. These targeting vectors were constructed to include a -23 kb human κ genomic sequence naturally located between human Vk4-1 and Jk1 gene segments. This human κ genomic sequence was specifically positioned in these two additional targeting vectors between human VA and human Jλ gene segments (FIG. 4B, 12(k)1-k and 12(k)4-k Targeting Vectors).

[0213] Both targeting vectors containing the human κ genomic sequence were made using the modified RP11-729g4 BAC clone described above (FIG. 6). This modified BAC clone was targeted with a spectinomycin selection cassette flanked by Notl and AsiSI restriction sites (FIG.6, top left). Homologous recombination with the spectinomycin cassette resulted in a double-targeted 729g4 BAC clone which included, from 5' to 3', an l-Ceul site, a 5' Frt site, a neomycin cassette, a 3' Frt site, a -123 kb fragment containing the first 12 hVA gene segments, a Notl site about 200 bp downstream (3') to the nonamer sequence of the hVA3-1 gene segment, a spectinomycin cassette and an AsiSI site. A separate human BAC clone containing human κ sequence (CTD-2366j12) was targeted two independent times to engineer restriction sites at locations between IiVk4-1 and hJKl gene segments to allow for subsequent cloning of a -23 kb fragment for ligation with the hVA gene segments contained in the double targeted modified 729g4 BAC clone (FIG. 6, top right).

[0214] Briefly, the 2366j12 BAC clone is about 132 kb in size and contains hVK gene segments 1-6, 1-5, 2-4, 7-3, 5-2, 4-1, human κ genomic sequence down stream of the Vk gene segments, hJK gene segments 1-5, the hCK and about 20 kb of additional genomic sequence of the human κ locus. This clone was first targeted with a targeting vector containing a hygromycin cassette flanked by Frt sites and a Notl site downstream (3') of the 3' Frt site. The homology arms for this targeting vector contained human genomic sequence 5' and 3' of the Vk gene segments within the BAC clone such that upon homologous recombination with this targeting vector, the Vk gene segments were deleted and a Notl site was engineered -133 bp downstream of the hVK4-1 gene segment (FIG. 6, top right). This modified 2366j12 BAC clone was targeted independently with two targeting vectors at the 3' end to delete the hJK gene segments with a chloroamphenicol cassette that also contained either a hJKl gene segment, a Pl-Scel site and an AsiSI site or a human X genomic fragment containing four hJA gene segments (supra), a Pl-Scel site and an AsiSI site (FIG. 6, top right). The homology arms for these two similar targeting vectors contained sequence 5' and 3' of the hJK gene segments. Homologous recombination with these second targeting vectors and the modified 2366j12 BAC clone yielded a double-targeted 2366j12 clone which included, from 5' to 3', a 5' Frt site, a hygromycin cassette, a 3' Frt site, a Notl site, a 22,800 bp genomic fragment of the human κ locus containing the intergenic region between the Vk4-1 and Jk1 gene segments, either a hJA1 gene segment or a human λ genomic fragment containing hJA1, JA2, JA3 and JA7, a Pl-Scel site and a chloroamphenicol cassette (FIG. 6, top right). Two final targeting vectors to make the two additional modifications were achieved by two ligation steps using the double-targeted 729g4 and 2366j12 clones.

[0215] Double targeted 729g4 and 2366j12 clones were digested with Notl and AsiSI yielding one fragment containing the neomycin cassette and hVA gene segments and another fragment containing the -23 kb genomic fragment of the human κ locus containing the intergenic region between the Vk4-1 and Jk1 gene segments, either a hJA1 gene segment or a genomic fragment containing hJA1, JA2, JA3 and JA7 gene segments, the Pl-Scel site and the chloroamphenicol cassette, respectively. Ligation of these fragments generated two unique BAC clones containing from 5' to 3' the hVA gene segments, the human κ genomic sequence between the Vk4-1 and Jk1 gene segments, either a hJA1 gene segment or a genomic fragment containing hJA1, JA2, JA3 and JA7 gene segments, a Pl-Scel site and a chloroamphenicol cassette (FIG. 6, bottom). These new BAC clones were then digested with l-Ceul and Pl-Scel to release the unique fragments containing the upstream neomycin cassette and the contiguous human λ and κ sequences and ligated into a modified mouse BAC clone 302g12 which contained from 5' to 3' mouse genomic sequence 5' of the endogenous κ locus, an l-Ceul site, a 5' Frt site, a neomycin cassette, a 3' Frt site, hVA gene segments (3-12 to 3-1), a Notl site -200 bp downstream of VA3-1, -23 kb of human κ sequence naturally found between the human Vk4-1 and Jk1 gene segments, either a hJA1 gene segment or a genomic fragment containing hJA1, JA2, JA3 and JA7 gene segments, the mouse Εκί, the mouse Ck gene and Ek3' (FIG. 4, 12hAA-VKjK-hJA1 and 12hVA-VKjK-4hJA Targeting Vectors). Homologous recombination with both of these targeting vectors created two separate modified mouse κ light chain loci containing 12 hVA gene segments, human κ genomic sequence, and either one or four hJA gene segments operably linked to the endogenous mouse Ck gene which, upon recombination, leads to the formation of a chimeric human λ/mouse κ light chain.

Example III

Engineering Additional Human V1A Genes Segments Into a Human λ Light Chain Mini-Locus [0216] Additional hVA gene segments were added independently to each of the initial modifications described in Example 2 using similar targeting vectors and methods (FIG. 5A, +16-A Targeting Vector and FIG. 5B, +16-K Targeting Vector).

[0217] Introduction of 16 additional human VA gene segments. Upstream (5') homology arms used in constructing targeting vectors for adding 16 additional hvA gene segments to the modified light chain loci described in Example 2 contained mouse genomic sequence 5' of either the endogenous κ or A light chain loci. The 3' homology arms were the same for all targeting vectors and contained human genomic sequence overlapping with the 5' end of the human A sequence of the modifications as described in Example 2.

[0218] Briefly, two targeting vectors were engineered for introduction of 16 additional hVA gene segments to the modified mouse light chain loci described in Example 2 (FIG. 5Aand 5B, +16-A or +16-K Targeting Vector). A -172 kb DNAfragment from human BAC clone RP11-761I13 (Invitrogen) containing 21 consecutive hVA gene segments from cluster A was engineered with a 5' homology arm containing mouse genomic sequence 5' to either the endogenous κ or A light chain loci and a 3' homology arm containing human genomic A sequence. The 5' mouse κ or A homology arms used in these targeting constructs were the same 5' homology arms described in Example 2 (FIG. 5Aand 5B). The 3' homology arm included a 53,057 bp overlap of human genomic A sequence corresponding to the equivalent 5' end of the -123 kb fragment of human genomic A sequence described in Example 2. These two targeting vectors included, from 5' to 3', a 5' mouse homology arm containing either -23 kb of genomic sequence 5' of the endogenous mouse κ light chain locus or -24 kb of mouse genomic sequence 5' of the endogenous A light chain locus, a 5' Frt site, a hygromycin cassette, a 3' Frt site and 171,457 bp of human genomic A sequence containing 21 consecutive hVA gene segments, -53 kb of which overlaps with the 5' end of the human A sequence described in Example 3 and serves as the 3' homology arm for this targeting construct (FIG. 5Aand 5B, +16-A or +16-K Targeting Vectors). Homologous recombination with these targeting vectors created independently modified mouse κ and A light chain loci each containing 28 hVA gene segments and a hJA1 gene segment operably linked to endogenous mouse constant genes (Ck or CA2) which, upon recombination, leads to the formation of a chimeric light chain.

[0219] In a similar fashion, the +16-K Targeting Vector was also used to introduce the 16 additional hVA gene segments to the other initial modifications described in Example 2 that incorporated multiple hJA gene segments with and without an integrated human κ sequence (FIG. 4B). Homologous recombination with this targeting vector at the endogenous mouse κ locus containing the other initial modifications created mouse κ light chain loci containing 28 hVA gene segments and hJA1, 2, 3 and 7 gene segments with and without a human Vk-Jk genomic sequence operably linked to the endogenous mouse Ck gene which, upon recombination, leads to the formation of a chimeric λ-κ light chain.

[0220] Introduction of 12 additional human VA gene segments. Additional hVA gene segments were added independently to each of the modifications described above using similar targeting vectors and methods. The final locus structure resulting from homologous recombination with targeting vectors containing additional hVA gene segments are shown in FIG. 7A and 7B.

[0221] Briefly, a targeting vector was engineered for introduction of 12 additional hVA gene segments to the modified mouse κ and A light chain loci described above (FIG. 5Aand 5B, +12-A or 12-k Targeting Vectors). A 93,674 bp DNA fragment from human BAC clone RP11-22118 (Invitrogen) containing 12 consecutive hVA gene segments from cluster B was engineered with a 5' homology arm containing mouse genomic sequence 5' to either the endogenous mouse κ or A light chain loci and a 3' homology arm containing human genomic A sequence. The 5' homology arms used in this targeting construct were the same 5' homology arms used for the addition of 16 hVA gene segments described above (FIG. 5A and 5B). The 3' homology arm was made by engineering a Pl-Scel site -3431 bp 5' to the human VA3-29P gene segment contained in a 27,468 bp genomic fragment of human A sequence from BAC clone RP11-761113. This Pl-Scel site served as a ligation point to join the -94 kb fragment of additional human A sequence to the -27 kb fragment of human A sequence that overlaps with the 5' end of the human A sequence in the previous modification using the +16-A or +16-K Targeting Vectors (FIG. 5Aand 5B). These two targeting vectors included, from 5' to 3', a 5' homology arm containing either -23 kb of mouse genomic sequence 5' of the endogenous κ light chain locus or -24 kb of mouse genomic sequence 5' of the endogenous A light chain locus, a 5' Frt site, a neomycin cassette, a 3' Frt site and 121,188 bp of human genomic A sequence containing 16 hVA gene segments and a Pl-Scel site, -27 kb of which overlaps with the 5' end of the human A sequence from the insertion of 16 addition hVA gene segments and serves as the 3' homology arm for this targeting construct (FIG. 5Aand 5B, +12-A or 12-k Targeting Vectors). Homologous recombination with these targeting vectors independently created modified mouse κ and A light chain loci containing 40 hVA gene segments and human JA1 operably linked to the endogenous mouse constant genes (Ck or CA2) which, upon recombination, leads to the formation of a chimeric light chain (bottom of FIG.5A and 5B).

[0222] In a similar fashion, the +12-K Targeting Vector was also used to introduce the 12 additional hVA gene segments to the other initial modifications that incorporated multiple hJA gene segments with and without an integrated human κ sequence (FIG. 4B). Flomologous recombination with this targeting vector at the endogenous mouse κ locus containing the other modifications created a mouse κ light chain locus containing 40 hVA gene segments and hJA1, 2, 3 and 7 gene segments with and without a human Vk-Jk genomic sequence operably linked to the endogenous mouse Ck gene which, upon recombination, leads to the formation of a chimeric λ-κ light chain.

Example IV

Identification of targeted ES cells Bearing Human λ Light Chain Gene Segments [0223] Targeted BAC DNA made according to the foregoing Examples was used to electroporate mouse ES cells to create modified ES cells for generating chimeric mice that express human λ light chain gene segments. ES cells containing an insertion of unrearranged human λ light chain gene segments were identified by a quantitative TAQMAN® assay. Specific primers sets and probes were design for insertion of human λ sequences and associated selection cassettes (gain of allele, GOA), loss of endogenous mouse sequences and any selection cassettes (loss of allele, LOA) and retention of flanking mouse sequences (allele retention, AR). For each additional insertion of human λ sequences, additional primer sets and probes were used to confirm the presence of the additional human λ sequences as well as the previous primer sets and probes used to confirm retention of the previously targeted human sequences. Table 1 sets forth the primers and associated probes used in the quantitative PCR assays. Table 2 sets forth the combinations used for confirming the insertion of each section of human λ light chain gene segments in ES cell clones.

[0224] ES cells bearing the human λ light chain gene segments are optionally transfected with a construct that expresses FLP in order to remove the Frt'ed neomycin cassette introduced by the insertion of the targeting construct containing human VK5-52-VA1-40 gene segments (FIG. 5Aand 5B). The neomycin cassette may optionally be removed by breeding to mice that express FLP recombinase (e.g., US 6,774,279). Optionally, the neomycin cassette is retained in the mice.

Table 1

Example V

Generation of Mice Expressing Human λ Light Chains From an Endogenous Light Chain Locus [0225] Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1 ):91-99. VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing human A gene segments were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detected the presence of the unique human A gene segments (supra).

[0226] κ:λ light chain usage of mice bearing human λ light chain gene segments. Mice homozygous for each of three successive insertions of hVA gene segments with a single hJA gene segment (FIG. 5B) and mice homozygous for a first insertion of hVA gene segments with either a single hJA gene segment or four human Jλ gene segments including a human Vk-Jk genomic sequence (FIG. 4B) were analyzed for κ and A light chain expression in splenocytes using flow cytometry.

[0227] Briefly, spleens were harvested from groups of mice (ranging from three to seven animals per group) and grinded using glass slides. Following lysis of red blood cells (RBCs) with ACK lysis buffer (Lonza Walkersville), splenocytes were stained with fluorescent dye conjugated antibodies specific for mouse CD19 (Clone 1 D3; BD Biosciences), mouse CD3 (17A2; Biolegend), mouse IgK (187.1; BD Biosciences) and mouse IgA (RML-42; Biolegend). Data was acquired using a BD™ LSR II flow cytometer (BD Biosciences) and analyzed using FLOWJO™ software (Tree Star, Inc.). Table 3 sets forth the average percent values for B cells (CD19+), κ light chain (CD19+lgK+lgA')D and λ light chain (CD19+lgK'lgA+) expression observed in splenocytes from groups of animals bearing each genetic modification.

[0228] In a similar experiment, B cell contents of the splenic compartment from mice homozygous for a first insertion of 12 hVA and four hJA gene segments including a human Vk-Jk genomic sequence operably linked to the mouse Ck gene (bottom of FIG. 4B) and mice homozygous for 40 hVA and one hJA gene segment (bottom of FIG. 5B or top of FIG. 7B) were analyzed for IgK and

IgA expression using flow cytometry (as described above). FIG. 8A shows the IgA and IgA expression in CD19+ B cells for a representative mouse from each group. The number of CD19+ B cells per spleen was also recorded for each mouse (FIG. 8B).

[0229] In another experiment, B cell contents of the spleen and bone marrow compartments from mice homozygous for 40 hVA and four hJA gene segments including a human Vk-JkA genomic sequence operably linked to the mouse Ck gene (bottom of FIG. 7B) were analyzed for progression through B cell development using flow cytometry of various cell surface markers.

[0230] Briefly, two groups (N=3 each, 9-12 weeks old, male and female) of wild type and mice homozygous for 40 hVA and four hJA gene segments including a human Vk-Jk genomic sequence operably linked to the mouse Ck gene were sacrificed and spleens and bone marrow were harvested. Bone marrow was collected from femurs by flushing with complete RPMI medium (RPMI medium supplemented with fetal calf serum, sodium pyruvate, Hepes, 2-mercaptoethanol, non-essential amino acids, and gentamycin). RBCs from spleen and bone marrow preparations were lysed with ACK lysis buffer (Lonza Walkersville), followed by washing with complete RPMI medium. 1x106 cells were incubated with anti-mouse CD16/CD32 (2.4G2, BD Biosciences) on ice for 10 minutes, followed by labeling with a selected antibody panel for 30 min on ice.

[0231] Bone marrow panel: anti-mouse FITC-CD43 (1 B11, BioLegend), PE-ckit (2B8, BioLegend), PeCy7-lgM (11/41, eBioscience), PerCP-Cy5.5-lgD (11-26c.2a, BioLegend), APC- B220 (RA3-6B2, eBioscience), APC-PI7-CD19 (ID3, BD) and Pacific Blue-CD3 (17A2, BioLegend).

[0232] Bone marrow and spleen panel: anti-mouse FITC-lgK (187.1, BD), ΡΕ-IgA (RML-42, BioLegend), PeCy7-lgM (11/41 ebioscience), PerCP-Cy5.5-lgD (11-26c.2a, BioLegend), Pacific Blue-CD3 (17A2, BioLegend), APC- B220 (RA3-6B2, eBioscience), APC-PI7-CD19 (ID3, BD).

[0233] Following staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a FACSCANTOII™ flow cytometer (BD Biosciences) and analyzed with FLOWJO™ software (Tree Star, Inc.). FIGs. 9A- 9D show the results for the splenic compartment of one representative mouse from each group. FIGs. 10A- 10E show the results for the bone marrow compartment of one representative mouse from each group. Table 4 sets forth the average percent values for B cells (CD19+), κ light chain (CD19+lgK+lgA')D and λ light chain (CD19+lgK'lgA+) expression observed in splenocytes from groups of animals bearing various genetic modifications. Table 5 sets forth the average percent values for B cells (CD19+), mature B cells (B220hilgM+), immature B cells (B220intlgM+), immature B cells expressing κ light chain (B220intlgM+lgK+) and immature B cells expressing λ light chain (B220lntlgM+lgA+) observed in bone marrow of wild type and mice homozygous for 40 hVA and four hJA gene segments including a human Vk-Jk genomic sequence operably linked to the mouse Ck gene. This experiment was repeated with additional groups of the mice described above and demonstrated similar results (data not shown).

[0234] Human VA gene usage in mice bearing human λ light chain gene segments. Mice heterozygous for a first insertion of human λ sequences (hVA3-12 - hVA3-1 and hJA1, FIG. 5B) and homozygous for a third insertion of human λ sequences (hVA5-52 - hVA3-1 and hJA1, FIG. 5B) were analyzed for human λ light chain gene usage by reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.

[0235] Briefly, spleens were harvested and perfused with 10 mL RPMI-1640 (Sigma) with 5% HI-FBS in sterile disposable bags. Each bag containing a single spleen was then placed into a STOMACHER™ (Seward) and homogenized at a medium setting for 30 seconds. Homogenized spleens were filtered using a 0.7pm cell strainer and then pelleted with a centrifuge (1000 rpm for 10 minutes) and RBCs were lysed in BD PHARM LYSE™ (BD Biosciences) for three minutes. Splenocytes were diluted with RPMI-1640 and centrifuged again, followed by resuspension in 1 mL of PBS (Irvine Scientific). RNA was isolated from pelleted splenocytes using standard techniques known in the art.

[0236] RT-PCR was performed on splenocyte RNA using primers specific for human hN/λ gene segments and the mouse Ck gene (Table 6). PCR products were gel-purified and cloned into pCR2.1-TOPO TA vector (Invitrogen) and sequenced with primers M13 Forward (GTAAAACGAC GGCCAG; SEQ ID NO: 55) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:56) located within the vector at locations flanking the cloning site. Eighty-four total clones derived from the first and third insertions of human λ sequences were sequenced to determine hN/λ gene usage (Table 7). The nucleotide sequence of the hVA-hJA1-mCK junction for selected RT-PCR clones is shown in FIG. 11.

[0237] In a similar fashion, mice homozygous for a third insertion of human λ light chain gene sequences (/.e. 40 hN/λ gene segments and four hJA gene segments including a human Vk-Jk genomic sequence, bottom of FIG. 7B) operably linked to the endogenous mouse Ck gene were analyzed for human λ light chain gene usage by RT-PCR using RNA isolated from splenocytes (as described above). The human λ light chain gene segment usage for 26 selected RT-PCR clones are shown in Table 8. The nucleotide sequence of the hVA-hJA-mCK junction for selected RT-PCR clones is shown in FIG. 12.

[0238] In a similar fashion, mice homozygous for a first insertion of human λ light chain gene segments (12 hN/λ gene segments and hJA1, FIG. 4A& FIG. 5A) operably linked to the endogenous mouse CA2 gene were analyzed for human λ light chain gene usage by RT-PCR using RNA isolated from splenocytes (as described above). The primers specific for hVA gene segments (Table 6) were paired with one of two primers specific for the mouse CA2 gene; CA2-1 (SEQ ID NO: 104) or CA2-2 (SEQ ID NO: 105).

[0239] Multiple hN/λ gene segments rearranged to hA1 were observed from the RT-PCR clones from mice bearing human λ light chain gene segments at the endogenous mouse λ light chain locus. The nucleotide sequence of the hVA-hJA-mCA2 junction for selected RT-PCR clones is shown in FIG. 13.

[0240] FIG. 11 shows the sequence of the hVA-hJA1-mCK junction for RT-PCR clones from mice bearing a first and third insertion of hVA gene segments with a single hJA gene segment. The sequences shown in FIG. 11 illustrate unique rearrangements involving different hVA gene segments with hJA1 recombined to the mouse Ck gene. Fleterozygous mice bearing a single modified endogenous κ locus containing 12 hVA gene segments and hJA1 and homozygous mice bearing two modified endogenous κ loci containing 40 hVA gene segments and hJA1 were both able to produce human A gene segments operably linked to the mouse Ck gene and produce B cells that expressed human A light chains. These rearrangements demonstrate that the chimeric loci were able to independently rearrange human A gene segments in multiple, independent B cells in these mice. Further, these modifications to the endogenous κ light chain locus did not render any of the hVA gene segments inoperable or prevent the chimeric locus from recombining multiple hVA and a hJA (JA1) gene segment during B cell development as evidenced by 16 different hVA gene segments that were observed to rearrange with hJA1 (Table 7). Further, these mice made functional antibodies containing rearranged human VA-JA gene segments operably linked to mouse Ck genes as part of the endogenous immunoglobulin light chain repertoire.

[0241] FIG. 12 shows the sequence of the hVA-hJA-mCK junction for selected RT-PCR clones from mice homozygous for 40 hVA and four hJA gene segments including a human Vk-Jk genomic sequence. The sequences shown in FIG. 12 illustrate additional unique rearrangements involving multiple different hVA gene segments, spanning the entire chimeric locus, with multiple different hJA gene segments rearranged and operably linked to the mouse Ck gene. Pbmozygous mice bearing modified endogenous κ loci containing 40 hVA and four hJA gene segments were also able to produce human A gene segments operably linked to the mouse Ck gene and produce B cells that expressed human A light chains. These rearrangements further demonstrate that the all stages of chimeric loci were able to independently rearrange human A gene segments in multiple, independent B cells in these mice. Further, these additional modifications to the endogenous κ light chain locus demonstrates that each insertion of human A gene segments did not render any of the hVA and/or JA gene segments inoperable or prevent the chimeric locus from recombining the hVA and JA gene segments during B cell development as evidenced by 12 different hVA gene segments that were observed to rearrange with all four hJA gene segments (Table 8) from the 26 selected RT-PCR clone. Further, these mice as well made functional antibodies containing human VA-JA gene segments operably linked to mouse Ck regions as part of the endogenous immunoglobulin light chain repertoire.

[0242] FIG. 13 shows the sequence of the hVA-hJA-mCA2 junction for three individual RT-PCR clones from mice homozygous for 12 hVA gene segments and hJA1. The sequences shown in FIG. 13 illustrate additional unique rearrangements involving different hVA gene segments, spanning the length of the first insertion, with hJA1 rearranged and operably linked to the mouse CA2 gene (2D1 = VA2-8JA1; 2D9 = VA3-10JA1; 3E15 = VA3-1JA1). One clone demonstrated a nonproductive rearrangement due to N additions at the hVA-hJA junction (2D1, FIG.13). This is not uncommon in V(D)J recombination, as the joining of gene segments during recombination has been shown to be imprecise. Although this clone represents an unproductive recombinant present in the light chain repertoire of these mice, this demonstrates that the genetic mechanism that contributes to junctional diversity among antibody genes is operating normally in these mice and leading to an antibody repertoire containing light chains with greater diversity.

[0243] Pbmozygous mice bearing modified endogenous A loci containing 12 hVA gene segments and hJA1 were also able to produce human A gene segments operably linked to an endogenous mouse CA gene and produce B cells that expressed reverse chimeric A light chains containing hVA regions linked to mouse CA regions. These rearrangements further demonstrate that human A light chain gene segments placed at the other light chain locus (/.e., the A locus) were able to independently rearrange human A gene segments in multiple, independent B cells in these mice. Further, the modifications to the endogenous A light chain locus demonstrate that the insertion of human A gene segments did not render any of the hVA and/or hJA1 gene segments inoperable or prevent the chimeric locus from recombining the hVA and hJA1 gene segments during B cell development. Further, these mice also made functional antibodies containing human VA-JA gene segments operably linked to a mouse CA region as part of the endogenous immunoglobulin light chain repertoire.

[0244] As shown in this Example, mice bearing human A light chain gene segments at the endogenous κ and A light chain loci are capable of rearranging human κλ light chain gene segments and expressing them in the context of a mouse Ck and/or CA region as part of the normal antibody repertoire of the mouse because a functional light chain is required at various checkpoints in B cell development in both the spleen and bone marrow. Further, early subsets of B cells (e.g., pre-, pro- and transitional B cells) demonstrate a normal phenotype in these mice as compared to wild type littermates (FIGs. 9D, 10Aand 10B). A small deficit in bone marrow and peripheral B cell populations was observed, which may be attributed to a deletion of a subset of auto-reactive immature B cells and/or a suboptimal association of human A light chain with mouse heavy chain. Fbwever, the IgK/lgA usage observed in these mice demonstrates a situation that is more like human light chain expression than that observed in mice.

Example VI

Breeding of Mice Expressing Human λ Light Chains From an Endogenous Light Chain Locus [0245] To optimize the usage of the human λ gene segments at an endogenous mouse light chain locus, mice bearing the unrearranged human λ gene segments are bred to another mouse containing a deletion in the opposing endogenous light chain locus (either κ or λ). For example, human λ gene segments positioned at the endogenous κ locus would be the only functional light chain gene segments present in a mouse that also carried a deletion in the endogenous λ light chain locus. In this manner, the progeny obtained would express only human λ light chains as described in the foregoing examples. Breeding is performed by standard techniques recognized in the art and, alternatively, by commercial companies, e.g., The Jackson Laboratory. Mouse strains bearing human λ light chain gene segments at the endogenous κ locus and a deletion of the endogenous λ light chain locus are screened for presence of the unique reverse-chimeric (human-mouse) λ light chains and absence of endogenous mouse λ light chains.

[0246] Mice bearing an unrearranged human λ light chain locus are also bred with mice that contain a replacement of the endogenous mouse heavy chain variable gene locus with the human heavy chain variable gene locus (see US 6,596,541, Regeneron Pharmaceuticals, the VELOCIMMUNE® genetically engineered mouse). The VELOCIMMUNE® mouse includes, in part, having a genome comprising human heavy chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies comprising a human heavy chain variable region and a mouse heavy chain constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy chains of the antibodies can be isolated and operably linked to DNA encoding the human heavy chain constant regions. The DNA can then be expressed in a cell capable of expressing the fully human heavy chain of the antibody. Upon a suitable breeding schedule, mice bearing a replacement of the endogenous mouse heavy chain locus with the human heavy chain locus and an unrearranged human λ light chain locus at the endogenous κ light chain locus is obtained. Antibodies containing somatically mutated human heavy chain variable regions and human λ light chain variable regions can be isolated upon immunization with an antigen of interest.

Example VII

Generation of Antibodies From Mice Expressing Human Heavy Chains and Human λ Light Chains [0247] After breeding mice that contain the unrearranged human λ light chain locus to various desired strains containing modifications and deletions of other endogenous Ig loci (as described above), selected mice are immunized with an antigen of interest.

[0248] Generally, a VELOCIMMUNE® mouse containing one of the single rearranged human germline light chain regions is challenged with an antigen, and lymphatic cells (such as B-cells) are recovered from serum of the animals. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies containing human heavy chain and human λ light chain that are specific to the antigen used for immunization. DNA encoding the variable regions of the heavy chains and the λ light chains may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Due to the presence of the additional hVA gene segments as compared to the endogenous mouse λ locus, the diversity of the light chain repertoire is dramatically increased and confers higher diversity on the antigen-specific repertoire upon immunization. The resulting cloned antibody sequences may be subsequently produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes (e.g., B cells).

[0249] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described above, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody containing a somatically mutated human heavy chain and a human λ light chain derived from an unrearranged human λ light chain locus of the invention. Suitable human constant regions include, for example wild type or modified lgG1, lgG2, lgG3, or lgG4.

SEQUENCE LISTING

[0250] <110> Macdonald, Lynn Stevens, Sean Gurer, Cagan Murphy, Andrew J. Hosiawa, Karolina A.

<120> HUMAN LAMBDA LIGHT CHAIN MICE

<130> 796A-WO <140> To be assigned <141 > <150> 61/357,314 <151 >2010-06-22 <150> 61/357,317 <151 >2010-06-22 <160> 105 <170> FastSEQ for Windows Version 4.0

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<210> 34 <211 > 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 34 gggctacttg aggaccttgc t 21

<210> 35 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 35 gacagccctt acagagtttg gaa 23

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<210> 38 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 38 cagggcctcc atcccaggca 20 <210> 39

<211> 28 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 39 ccccagtgtg tgaatcactc taccctcc 28

<210> 40 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 40 cctctcctcc tcaccctcct 20

<210>41 <211> 20 <212> DNA <213> artificial sequence <220> <221 > variation <222> (4)...(4) <223> r=a or g <220> <221 > variation <222> (9)...(9) <223> s=c or g <220> <221 > variation <222> 11, 12, 13 <223> y=c or t <400> 41 atgrccdgst yyyctctcct 20

<210> 42 <211 > 18 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 42 ctcctcactc agggcaca 18

<210> 43 <211> 20 <212> DNA <213> artificial sequence <220> <221 > variation <222> (18)...(18) <223> s=c or g <400> 43 atggcctggg ctctgctsct 20

<210> 44 <211 > 19 <212> DNA <213> artificial sequence <220> <221 > variation <222> (11)...(11) <223> y=c or t <220> <221 > variation <222> (13)...(13) <223> s=c or g <400> 44 atggcctgga ycsctctcc 19

<210> 45 <211> 23 <212> DNA <213> artificial sequence <220> <221 > variation <222> 11, 16, 18, 21 <223> y=c or t <220> <221 > variation <222> (15)...(15) <223> r=a or g <220> <221 > variation <222> (20)...(20) <223> m=a or c <400> 45 tcaccatggc ytggrycycm ytc 23

<210> 46 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 46 tcaccatggc ctgggtctcc tt 22

<210> 47 <211> 22 <212> DNA <213> artificial sequence <220> <221 > variation <222> (16)...(16) <223> m=a or c <220> <221 > variation <222> (19)...(19) <223> y=c or t <400> 47 tcaccatggc ctggamtcyt ct 22

<210> 48 <211> 26 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 48 tcaccatggc ctgggctcca ctactt 26

<210> 49 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 49 tcaccatggc ctggactcct 20

<210> 50 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 50 tcaccatggc ctggatgatg ctt 23

<210>51 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 51 taaatatggc ctgggctcct ct 22

<210> 52 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 52 tcaccatgcc ctgggctctg ct 22

<210> 53 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 53 tcaccatggc cctgactcct ct 22

<210> 54 <211> 30 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 54 cccaagctta ctggatggtg ggaagatgga 30

<210> 55 <211> 16 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 55 gtaaaacgac ggccag 16

<210> 56 <211 > 17 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 56 caggaaacag ctatgac 17

<210> 57 <211 >440 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 57 gggcctgggc tctgctgctc ctcaccctcc tcactcaggg cacagggtcc tgggcccagt 60 ctgccctgac tcagcctccc tccgcgtccg ggtctcctgg acagtcagtc accatctcct 120 gcactggaac cagcagtgac gttggtggtt ataactatgt ctcctggtac caacagcacc 180 caggcaaagc ccccaaactc atgatttatg aggtcagtaa gcggccctca ggggtccctg 240 atcgcttctc tggctccaag tctggcaaca cggcctccct gaccgtctct gggctccagg 300 ctgaggatga ggctgattat tactgcagct catatgcagg cagcaacaat ttcgtcttcg 360 gaactgggac caaggtcacc gtcctagggg ctgatgctgc accaactgta tccatcttcc 420 caccatccag taagcttggg 440

<210> 58 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 58 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt. tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcacta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441

<210> 59 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 59 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441

<210> 60 <211 > 438 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 60 atggcctggg ctctgctcct caccctcctc actcagggca cagggtcctg ggcccagtct 60 gccctgactc agcctccctc cgcgtccggg tctcctggac agtcagtcac catctcctgc 120 actggaacca gcagtgacgt tggtggttat aactatgtct cctggtacca acagcaccca 180 ggcaaagccc ccaaactcat gatttatgag gtcagtaagc ggccctcagg ggtccctgat 240 cgcttctctg gctccaagtc tggcaacacg gcctccctga ccgtctctgg gctccaggct 300 gaggatgagg ctgattatta ctgcagctca tatgcaggca gcaacaatta tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatccagta agcttggg 438

<210>61 <211 > 438 <212> DNA <213> artificial sequence <220 <223> synthetic <400 61 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatccagta agcttggg 438

<210> 62 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 62 atggcctggg ctctgctcct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacag.tcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441

<210> 63 <211 > 442 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 63 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa tttatgtctt 360 cggaactggg accaaggtca ccgtcctagg ggctgatgct gcaccaactg tatccatctt 420 cccaccatcc agtaagcttg gg 442

<210> 64 <211 > 428 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 64 ccttcatttt ctccacaggt ctctgtgctc tgcctgtgct gactcagccc ccgtctgcat 60 ctgccttgct gggagcctcg atcaagctca cctgcaccct aagcagtgag cacagcacct 120 acaccatcga atggtatcaa cagagaccag ggaggtcccc ccagtatata atgaaggtta 180 agagtgatgg cagccacagc aagggggacg ggatccccga tcgcttcatg ggctccagtt 240 ctggggctga ccgctacctc accttctcca acctccagtc tgacgatgag gctgagtatc 300 actgtggaga gagccacacg attgatggcc aagtcggttg tgtcttcgga actgggacca 360 aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca ccatccagta 420 agcttggg 428

<210> 65 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 65 atgacctgct cccctctcct cctcaccctt ctcattcact gcacagggtc ctgggcccag 60 tctgtgttga cgcagccgcc ctcagtgtct gcggccccag gacagaaggt caccatctcc 120 tgctctggaa gcagctccaa cattgggaat aattatgtat cctggtacca gcagctccca 180 ggaacagccc ccaaactcct catttatgac aataataagc gaccctcagg gattcctgac 240 cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact 300 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtgagcagtt a 441 <210> 66 <211> 441

<212> DNA <213> artificial sequence <220> <223> synthetic <400> 66 atgacctgct cccctctcct cctcaccctt ctcattcact gcacagggtc ctgggcccag 60 tctgtgttga cgcagccgcc ctcagtgtct gcggccccag gacagaaggt caccatctcc 120 tgctctggaa gcagctccaa cattgggaat aattatgtat cctggtacca gcagctccca 180 ggaacagccc ccaaactcct catttatgac aataataagc gaccctcagg gattcctgac 240 cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact 300 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc ggcttttttt 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtgagcagtt a 441

<210> 67 <211 > 345 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 67 cccgggcaga gggtcaccat ctcttgttct ggaagcagct ccaacatcgg aagtaatact 60 gtaaactggt accagcagct cccaggaacg gcccccaaac tcctcatcta tagtaataat 120 cagcggccct caggggtccc tgaccgattc tctggctcca agtctggcac ctcagcctcc 180 ctggccatca gtgggctcca gt.ctgaggat gaggctgatt attactgtgc agcatgggat 240 gacagcctga atggttatgt cttcggaact gggaccaagg tcaccgtcct aggggctgat 300 gctgcaccaa ctgtatccat cttcccacca tccagtgagc agtta 345

<210> 68 <211 > 432 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 68 atggcctgga cccctctcct gctccccctc ctcactttct gcacagtctc tgaggcctcc 60 tatgagctga cacagccacc ctcggtgtca gtgtccccag gacaaacggc caggatcacc 120 tgctctggag atgcattgcc aaaaaaatat gcttattggt accagcagaa gtcaggccag 180 gcccctgtgc tggtcatcta tgaggacagc aaacgaccct ccgggatccc tgagagattc 240 tctggctcca gctcagggac aatggccacc ttgactatca gtggggccca ggtggaggat 300 gaagctgact actactgtta ctcaacagac tacagtggta atcatgtctt cggaactggg 360 accaaggtca ccgtcctagg ggctgatgct gcaccaactg tatccatctt cccaccatcc 420 agtgagcagt ta 432

<210> 69 <211 > 426 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 69 atggcctgga ctcctctctt tctgttcctc ctcacttgct gcccagggtc caattcccag 60 gctgtggtga ctcaggagcc ctcactgact gtgtccccag gagggacagt cactctcacc 120 tgtggctcca gcactggagc tgtcaccagt ggtcattatc cctactggtt ccagcagaag 180 cctggccaag cccccaggac actgatttat gatacaagca acaaacactc ctggacacct 240 gcccggttct caggctccct ccttggggg.c aaagctgccc tgaccctttc gggtgcgcag 300 cctgaggatg aggctgagta ttactgcttg ctctcctata gtggtgctta tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatcc 426

<210> 70 <211> 331 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 70 agtggtcctg ggacagacgg ccaggattac ctgtggggga aacaacattg gaagtaaaaa 60 tgtgcactgg taccagcaga agccaggcca ggcccctgtg ctggtcatct atagggataa 120 caaccggccc tctgggatcc ctgagcgatt ctctggctcc aactcgggga acacggccac 180 cctgaccatc agcagagccc aagccgggga tgaggctgac tattactgtc aggtgtggga 240 cagcagcact tatgtcttcg gaactgggac caaggtcacc gtcctagggg ctgatgctgc 300 accaactgta tccatcttcc caccatccag t 331

<210>71 <211 > 417 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 71 actcctctcc tcctcctgtt cctctctcac tgcacaggtt ccctctcgca ggctgtgctg 60 actcagccgt cttccctctc tgcatctcct ggagcatcag ccagtctcac ctgcaccttg 120 cgcagtggca tcaatgttgg tacctacagg atatactggt accagcagaa gccagggagt 180 cctccccagt atctcctgag gtacaaatca gactcagata agcagcaggg ctctggagtc 240 cccagccgct tctctggatc caaagatgct tcggccaatg cagggatttt actcatctct 300 gggctccagt ctgaggatga ggctgactat tactgtatga tttggcacag cagcgcttat 360 gtcttcggaa ctgggaccaa ggtcaccgtc ctaggggctg atgctgcacc aactgta 417

<210> 72 <211 > 393 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 72 tttctgttcc tcctcacttg ctgcccaggg tccaattctc agactgtggt gactcaggag 60 ccctcactga ctgtgtcccc aggagggaca gtcactctca cctgtgcttc cagcactgga 120 gcagtcacca gtggttacta tccaaactgg ttccagcaga aacctggaca agcacccagg 180 gcactgattt atagtacaag caacaaacgc tcctggaccc ctgcccggtt ctcaggctcc 240 ctccttgggg gcaaagctgc cctgacactg tcaggtgtgc agcctgagga cgaggctgag 300 tattactgcc tgctctacta tggtggtgct tatgtcttcg gaactgggac caaggtcacc 360 gtcctagggg ctgatgctgc accaactgta tcc 393

<210> 73 <211 > 417 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 73 atggcctggg ctctgctgct cctcactctc ctcactcagg acacagggtc ctgggcccag 60 tctgccctga ctcagcctgc ctccgtgtct gggtctcctg gacagtcgat caccatctcc 120 tgcactggaa ccagcagtga tgttgggagt tataaccttg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gagggcagta agcggccctc aggggtttct 240 aatcgcttct ctggctccaa gtctggcaac acggcctccc tgacaatctc tgggctccag 300 gctgaggacg aggctgatta ttactgctgc tcatatgcag gtagtagcac ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatc 417

<210> 74 <211> 348 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 74 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cacttatgtc 300 ttcggaactg ggaccaaggt caccggcctg ggggctgatg ctgcacca 348

<210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 75 aacaaccgag ctccaggtgt 20

<210> 76 <211 > 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 76 agggcagcct tgtctccaa 19

<210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 77 cctgccagat tctcaggctc 20

<210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 78 catcacaggg gcacagactg 20

<210> 79 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 79 gatttgctga gggcagggt 19

<210> 80 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 80 ccccaagtct gatccttcct t 21

<210>81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 81 gctgaccaac gatcgcctaa 20

<210> 82 <211 > 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 82 taagcgccac actgcacct 19

<210> 83 <211 > 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 83 cctgccagat tctcaggctc cctg 24

<210> 84 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 84 ctgattggag acaaggctgc cct 23

<210> 85 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 85 ccttcatact cttgcatcct cccttctcca 30 <210> 86

<211> 35 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 86 ttccttctct tctgtgactc aattatttgt ggaca 35

<210> 87 <211 > 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 87 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttctgtgt tcggaggagg cacccggctg 120 accgccctcg gggctgatgc tgcaccaact gtatccatc 159

<210> 88 <211 > 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 88 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttatgtct tcggaactgg gaccaaggtc 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159

<210> 89 <211 > 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 89 tctggcacct cagcctccct ggccatcagt gggctccagt ctgaggatga ggctgattat 60 tactgtgcag catgggatga cagcctgaat ggtgctgtgt tcggaggagg cacccagctg 120 accgccctcg gggctgatgc tgcaccaact gtatccatc 159

<210> 90 <211 > 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 90 tctggcacct cagcctccct ggccatcagt gggctccggt ccgaggatga ggctgattat 60 tactgtgcag catgggatga cagcctgagt ggtcgggtgt tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159

<210> 91 <211> 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 91 tcggggaaca cggccaccct gaccatcagc agagcccaag ccggggatga ggctgactat 60 tactgtcagg tgtgggacag cagcåctgct gtgttcggag g'aggcaccca gctgaccgcc 120 ctcggggctg atgctgcacc aactgtatcc atc 153

<210> 92 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 92 tcagggacaa tggccacctt gactatcagt ggggcccagg tggaggatga agctgactac 60 tactgttact caacagacag cagtggtaat gctgtgttcg gaggaggcac ccagctgacc 120 gccctcgggg ctgatgctgc accaactgta tccatc 156

<210> 93 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 93 tcagggacaa tggccacctt gactatcagt ggggcccagg tggaggatga agctgactac 60 tactgttact caacagacag cagtggtaat catagggtgt tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159

<210> 94 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 94 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttatgtct tcggaactgg gaccaaggtc 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159

<210> 95 <211 > 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 95 gatgcttcgg ccaatgcagg gattttactc atctctgggc tccagtctga ggatgaggct 60 gactattact gtatgatttg gcacagcagc gctgtggtat tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159

<210> 96 <211 > 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 96 cttgggggca aagctgccct gacactgtca ggtgtgcagc ctgaggacga ggctgagtat 60 tactgcctgc tctactatgg tggtgctcgg gtgttcggcg gagggaccaa gctgaccgtc 120 ctaggggctg atgctgcacc aactgtatcc atc 153

<210> 97 <211 > 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 97 cttgggggca aagctgccct gaccctttcg ggtgcgcagc ctgaggatga ggctgagtat 60 tactgcttgc tctcctatag tggtgctcga gtattcggcg gagggaccaa gctgaccgtc 120 ctaggggctg atgctgcacc aactgtatcc atc 153

<210> 98 <211 > 165 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 98 tcaggcctga atcggtacct gaccatcaag aacatccagg aagaggatga gagtgactac 60 cactgtgggg cagaccatgg cagtgggagc aacttcgtgt ctgtgttcgg aggaggcacc 120 cagctgaCcg ccctcggggc tgatgctgca ccaactgtat ccatc 165

<210> 99 <211> 164 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 99 tctggcacgt cagccaccct gggcatcacc ggactccaga ctggggacga ggccgattat 60 tactgcggaa catgggatag cagcctgagt gctggccccg ggtgttcggc ggagggacca 120 agctgaccgt cctaggggct gstgctgcac caactgtatc catc 164

<210> 100 <211 >22800 <212> DNA <213> Artificial Sequence <220> <223> synthetic <900>100 aagctctaaa actacaaact gctgaaagat ctaatgacta ggacagccta gtaattttca 60 taggggcata aatgtgaaac gccttgtgca tcgtagaaga aagcagaaga gaaagcattc 120 ccaatttctt aactgccttt tacctatatt aatcagtaat atactggctt ttacctctgt 180 taatcataat aaacaaattc tcaataaatt ttatcgatac tcttcaatgc ctgctcagca 240 acattttccg aaggcagctc aagatattaa ataactcata agggccaacc tcctattgca 300 gcattctttg ggatttaacc agtttcccaa gactcttttc acaatgttaa gatgttagaa 360 atagatccaa aactaggtga tatatcccct agtaaaactg tgaggtcaaa cttgtctggc 420 taatgcttcc atttaaaaat ttctctttct tgatccttca ttgtatgtac acaataaatc 480 aggggaaaac tttaactgag tgaatcaaag tattctcatt attataatag gagcttcaca 540 cacacacaaa aaaatcaatt ctattactct cagcctcagt tcctaaagcc aagttaaagt 600 cctgttctaa gatcattgtt gcatgaccat atgtattcca ggtctaatct aaactgtgga 660 taaatcccag caggacatta gagatttttg tgagagtaag catataggat tcagggttta 720 tgagcettag atttttcttg tcaaaatgaa tgagagttgc catatctaaa aattattccc 780 agataaataa aattcactac ctagaattaa tttatgcata taagtagaaa tgctatctcc 840 ctttttacca tccaaagtgg aaagcctcat ggaactagaa attaatatta gaaaaatcag 900 ttaataaaag tatgtcattt catcaattca ataagttata atagcaaaaa accataataa 960 attatcactt aaatgtcaat acatttataa actatggtac ataaatagga tattgaatag 1020 ccattgatgc tcctgatgaa aattagcagg cagtgataaa tgataaatat gaagcacatg 1080 tcaataaata aaataagttt tatgtaattt aggagaaaat ggtgataatg acacaaaatg 1140 tgaattatgg atgcatctat aaaattcttt gtacatttgt gaattgtaaa tatttatctt 1200 agagacatta ttactttgta tatgttccat ttgctcacct atatgtccca gtctccttac 1260 aaatgctatg gccaaagaaa taggcataca tacatccttt gcaggctgag gcaggaaaaa 1320 gatcttacgg aattttccag tctatccttt atctgtataa gcaacttaag aggccatgtg 1380 ctccaaatgg tgcaaataca agatggtaga gcctctgtct gcctggatcc ttgagtggct 1440 gcatggagca gagcaccttt ctggccctgg tgaagattgt agcatgagca agatataagc 1500 atttgttgga gctaggccat gagatttggg gcagtggtat aacctaccct attatggaaa 1560 atataaatac acaaaacaga aaagagagag agaagtgaga gaagactgtg agagaagtgc 1620 atgagagaag actgtgtttt gttcatttcc tataatccta tatcaccatg ggatcctgtg 1680 ccttctggtg atcaaactaa tgttctacag ctccaaagaa gaatgctcgc otaacgtctc 1740 cattccaatg acctagagac taaaagccaa aaagaacctt agaaattatc tattgcattc 1800 tttgatgtaa ggaaatatct tagagggcac agatagaaat atcttaaccc aggtcactta I860 gttcgtggca gagctgaggc taaaaccagg ccttttgact cctaattttg tgctctttac 1920 accttctcac atcacttctc caacccaaag tctagcagaa aaggctaaaa taagatatat I960 gcatagattt gctattataa gtccatgtac ttcctcagac gctttaagat ggggcttctc 2040 atiggttcaca ataagcagca gagggaagtg aataactatc ttcgtctccc ctactgctat 2100 ttgtgcagtt tgaagcttat ctcttaaatic atgttttctt cLcgtagtaa atactacaac 2160 ttgtgccttt tatgtgtgta taaattttaa tataattttt ttccatgaac cattcaagta 2220 aaatggacac tccaaaaaga tgttcaataa ggttacatgg cttcacattg ccccctctac 2280 accatcttgt ggagctacac attcacctca cccaaatttg agaaaaataa tcaagaaaat 2340 gactctcact agcagtgaga ccaagtccat aagcactaat gtcatcagtg cacactgcag 2400 cctcatgctg ccaagcatgt tttgggcgta tccctggact ggtttggtga catgatcaaa 2460 ggtacatttt ccacctgcat agccccatcc tggatctata gccttccttg tgtctttgtg 2520 aacaacctag tgtgaactca aagtatgaga cagatctcaa ttaatttaga aagtttattt 2580 tcccaagatt aaggacaagc ccatgataaa gcctccagag gtcctgatat atgtgcccaa 2640 gggggtcggg gcacagcttg gtgttataca ttttagggag acaagaaaca tcaatcgata 2700 tgtagaagat gtgcatcgct ttggtctgga aaggtgtgac aactcaaggc agggaagggg 2760 gcttcctgct ggggttgcat tgttttgagt ctctgatcag cctttcacat gtgaaaggca 2820 ggtagagaaa tagtcattta tgccttagtc tggcttattg aaacagtagg gcagaagaag 2880 cattgcatat gcatttgtct gaagtgaaca gagggatgac tttgagctct gtcctttctt 2940 tgtccacaag gaattacctt gtgggcaaat tgtgagggag gtatgtagct. tttttttctt 3000 tgtagctatc ttatttagga ataaaatggg aggcaggttt gcctgatgca attcccagct 3060 tgactttccc ttttggctta gtgatttttg gggtcctgag gtttatttct tctttcacat 3120 tagtataact acttttcttt ttctaattcc ttttctactt gtatgtgtta cagctgactt 3180 atgttacttg caaaaagaat tctgactaat gcaccatctg actagaaggc agggtCcttc 3240 gatgataacg aatcctccag aatctagtaa acagaattgc ctgaaaaaga ggtgggtgtc 3300 ttcttgggga atttctcatg gcaatgaatg gcaactggcc aaaggattta tgaccagact 3360 gagctctctt ttatctattc tgttactcac caagacctat tagggtttgt gctccacagg 3420 gacactggtt tctaagttct agggttaaac agtccactcc caggcccacc acaccatacc 3480 ctcctgacat ctggtgaaca gcaataaaat tgtttcttat tctgaaaatc ctccaatact 3540 tccaccatcc ccaaaaatgc agtggaggag gagagaaaat gaattgttcc attagagaac 3600 acaatatccs ttatattatt cttggccttt gagatacctt acaaaacaaa tacaaaaaaa 3660 gtcccaattt aacatctttt aataatcttt acaaaacaga acacatctcc tttcttgata 3720 atagtcaaga ggctcagtgg caactgtggt gaaaagtgtc agattctggt catgtttcaa 3780 aggtagaaaa aatagaattt gttaacatat tggatgtgag gcgtgggaga aacgtgaaat 3840 caaggtggtt gcaagtgttt aacctgagca actagagaat ttggaaggac attttctgag 3900 atggggaagg caggcgggaa tcagggatta gagttgaaca tattagacat ttgagatgcc 3960 tgctagacct ctaattggca atatcccttg gacaggtgga tgaatatgcg tgattctgga 4020 gtt'cgggaea tagtccgggt ggagatgcaa atttgggaaa cagggcgagg ttactagcaa 4080 tgagttaaat caatgaaggc aggctgggac ctggcaggta acccaacaag tagaggtcga 4140 agagatgaga agaaaacagc acaggagact tagaagcagt ggtcaggagg aaggagttga 4200 accaagaaag tgatgtccca gagccaacaa aataaggatt tcctttctgt ttacaaatgt 4260 aaaattaaaa ggtttaataa aaagaaaatt tacttttatg gttggttgtt attaagtggt 4320 ccaaacactc tctcctattt gtagaatcag aactctctca tggcagtaga aaatttggaa 4380 agttactttt taaaaggtgt gtgcactgct gccctttgct ggtcaagttt atgcactgca 4440 aattccaagg acgattgctc gtcagctttt ctcctttaaa atagctcagg ctgtacaagc 4500 tagaaagaac ctcgcaagat attccttcca acatttgcat ttgacttatg ggaagtgcag 4560 gttcagccag aaaagttgtg tgcaaggccg tttatgtaag tttatcagac ctgattctta 4620 cggctcttcc cattgtttcg agcctccctt ccattcactt cccgctcata cgcgaccaag 4680 tataggacag gagtagttat tctgcacttt atagcagctc cactgtctgg cactctgatg 4740 ttctttsatt acaagcttta tgacagtgat tctcaacctg ctccactgcc tccacctagt 4800 ggcagaaaga agaaaatgtg tgtaactcgg gagtctctgg tctgaaagct ccggggtatc 4860 atttcttcaa agtcttgagc ttgtttttgt ttgtatttat ttatttattt gttttagaga 4920 caaggtctcg cactgcactc cagcctggga gacagagcga gacaattcag gatctatcta 4980 gtgaataaag agatatcagt aatgactgtt ttatattgtg gctgtagcgc attcgaggga 5040 taattcgatt ctgttctgct ttcgaatgca tggctcactg taacctccaa ctcccgggct 5100 caagcgatcc tcctacctca gcttctccag tagttgagct tgatttattt taaagtttca 5160 taaaattttg gcatttcttt ccacaatatg gccatgtgtg ctttactata aaatattttc 5220 atcacaaaat ttacatcgct ggaaatcccc ataagccagt ttgagaaaca caacccaaga 5280 aagcagaaca gactcaaatt atcccttaaa tcccccttaa ccacaaatat aaaacagtcc 5340 gtgactgggc gtgttggctt acacctgtaa tcccagcact ttgggaggcc aaggcgggtg 5400 gattacttga gctcaggagt tcaagaccag cctggccaac atggtgaaac cccgtcccta 5460 ttaaaaatac aaaattattc aggagttgtg gcaggcagtt gtaatcccag ctacttggga 5520 ggctgaggca ggagaatcac ttgaacccag gaggtggagg ttgtagtgag ccaagattgt 5580 gccagtgcac tccagcctgg gcaacagagc gagacttcca tcttaaaaaa aaaaaattaa 5640 gtaaataaaa tataaaaaaa taaagcagtc cctattgata tctctttatt cactaaatca 5700 acctggaatt gacctgaatt ctgatttttt tttcatcatg gattttttgc attaattttg 5760 attgtttaaa tattgcatta aaatattatt tatcttgact actgagtttg cgggacctcc 5820 ttaaaattta tgaccaaggc aatgcctcac tcactcgcct taccataatc tgggccacat 5880 atcaggggct ccaatagcaa gcaacatgac ttttgaacag ctaagacttc tctcttcact 5940 gtgaagacca gatgggccct gcaaacagtg taacctctac atgaaaatgc acgagattcc 6000 aactacaacc aggcacaaaa gactctgatg gtgaagtccc agccctccaa gtcccaactt 6060 cctgaaggga aagagcaccc caagttct'ga ccagaggcca gagtcataac gaagatggaa 6120 tgtgagcttg acatagaagg ggtggtagca cctggctcag taatgaagag gctttcggtc 6180 ctgaaggaag agctcagcac attcaaagat tagaagggag gtcccagtca taggagcagg 6240 gaaggagaga aggcccaata agaaacacag acaggaggga ggggtcaggg caagatcata 6300 ctggaaacaa ctagagagct aataaaagtc acagtgccca gtccccacat ggaccagact 6360 cttcggaatc tctaggcatc aatttgggca ccagtagttt tcaaagttct ccagaagatt 6420 ctatgcacac cagccaaggg tgggaaccac aggtgttggc ctagggatca tgacaatgag 6480 tttctaagtg caataagaaa cctccagaga gtttaagcag gggaataatt tgatttgttt 6540 cttgtttgtg atttttaaag atcagtctgg ttactgtgtg taagacaata atccagaaaa 6600 tctgttgctc atgaaccaca tatctgtaaa tttgcttccc ctgtaactgg atctaaccaa 6660 caaaaattag tacttactaa gaaattacat gcccagggac tatgctaagt aattcataaa 6720 cactatttta tttactcctc acagcaagtt tataagagaa acgttattat ttccacattt 6780 cggatgagaa atttgaggct tggggaaagt taagtaattt acctaatgcc acacccagtt 6840 cataagatgc agagttaaga ttctaattct gtgtctaagt tgatgctcca tcaaacacac 6900 cacgcctcca actaggaagc aacatgctgg ccagaggatg ctgtcatcaa gtttacagaa 6960 tggttagatt tctaggcaca gatgaataaa tcaacatgtt ggtttgcaat agaatgaatc 7020 tatccagctc tgaatttgca tccaagggtt tgtgagcaca caagtctaaa agtgtggcct 7080 cagctctgct aacttcatca aggtgaatac ctaggaggcc accctctgag accaccagat 7140 ggacagtcca ccatctgttt acagatggta aagccacata ccagctttgc catctgatgt 7200 tctctattca cattcaacat ttatacaaga aatagtcata tggatccttt tcaatagaca 7260 gtactgggga aattgaattg ccatatgcag aagaatggaa ctagacctct atctctcacc 7320 aaatacaaaa gttaactcaa gacagattaa agacttacat ataagacctg taactacaaa 7380 aacactagaa gaaaacctag ggaaaatgct tctggaatta atctaggtga agaactcagg 7440 actaagatat caaaagcaca agcaccaaaa caaaaataga caaacaggac ttaattaaac 7500 tagaacgctt ctgaacagca agagaaataa tcaatagagt gaacagataa tctgcagaat 7560 gggtgaaaat atttgcaaac tatgcatcct acagggaaat aatgtccaga atttagaagg 7620 aactcaaaca attcaacaac aacagcaaaa taaccccacc aaaaaagtgg gcaaaggaca 7680 tgaatagaca tttctcaaaa gaaggtatat gatatggttt ggctctgtgt ctccacccag 7740 atctcacctt aaattgtaat aatccccaca tatcatggga gagacccggt gggaggtaat 7800 tgaatcatgg gggcaggttt gtcccatgct gttctcatga tactgaataa gtcctatgag 7860 atctgatgat tttataaagg ggagttcccc tgcacacact ctcttgcctg cctccatgta 7920 atatgtgcct ttgcttctcc tttgccttct gccatgattg tgaggcctct ccagccatat 7980 ggaactgagt caattaaacc actttttctt tgtaaattac ccaatcttgg gtatgtcttt 8040 attagcagca taagaacaga ctaatacagt gtacaaatgg ccaagaagcg tacaaaaaac 8100 aaaatgctca aatcactaat cactagagaa tcgcaagtta aaaccacaat gagatattat 8160 cttacagcag tcagaatgcc tattattaaa acaccaaaaa ataacatgtt ggcaaggatg 8220 cagagaaaag ggaatactta cacattatta gtgggaatgt aaactagtac agcttctgtg 8280 gaaaacacta tggagatttc tcaaagaact agaaatagaa ctaccatgtg gttcagcaat 8340 accacaactg ggtatctacc caaagggaaa taaattatta tataaaaaag atatctgcac 8400 tcacttgttt attgcagcac tattcacaat agcaaagata tggaatcaac ccaagtgtcc 8460 atcaacagat gattggataa agaaaacgtg gtgtgtgtgt gtgtgtgtgt gtgtgtgtat 8520 acacatacca caatgaaata ctattcagct ataaagaaaa gaatgaaatc atgtct~ttg 8580 cagcaatgtg gttggaactg gaggccatta tcttaagtgg ataattcaaa aacagaaggt 8640 caaatgtcac atgttctcac ttataagtgg gagctaaatg atgtgtacac atggacatag 8700 agtgtggtat gataaacact ggagattgag atgggtggaa gggtggaagg aggttgagtg 8760 atgagaaaat actaaatgga tacaatatac atgattcagg cgatagatac actaaaagcc 8820 cagacttcac cactacacag tatagctatg tagcaaaatt gcacctgtat tgcttaaatt 8880 tatacaagta aaaaaaagat cgtacgaatt ctgtttttta ttctctatga aattactact 8940 gagagtatta tccaatgccg tttctatgca gtgcccccaa tattatccat ttagcagctc 5000 ctatgcaatg ccccaagata gaaattgtct tcaactttta tcccaggaaa accttcagtc 9060 acacgtagaa actagaaatt tttcccctag atgaaagtta tgtaacataa cacattatct 9120 tcatttagtc ggtttccaag aagctcagaa ccagatttta tgttcaatca aaaectgctt 9180 attttaagtg aggtttactg aggtataaat tacaataaaa gccacctttt cgtgtatatt 9240 tctataagtt ttggcaaatg catagctgtg taaccacaac cacattcaag atataggaca 9300 agtccctcat octttaaagt tcctttatgc cccttccttc accccagccc ttggcaacca 9360 ctggtttttg tctgatccaa tcgtttgcct cttcctgaat gtcatgtaaa tagagccatg 9420 caatgtgaag ccttttgagt ctggctttgt tcacttgttc acttaggaga atgcatttqa 9480 gattcatctt tgctgtttcg tgtagcacta gttcactgtc tattgttgag tagtattcca 9540 ttgtgtggat atgccacaga ttgtttatct agttaacaat ttaaagccat ttggtcattt 9600 ctaattttta gctgctaaga ataaagttgc tgtaagcttt ccaatgcagg tttttgtgtg 9660 aactcaggat ttcatttcgc ttgggtaaat tcctagcttt gggactgctg agtcatctgg 9720 taggtgtatg ttgaacttta taagaaactg ccaaactgtt ttccaaagtt gctgtgctct 9780 tttgcactcc catcagcagt gaatgagggt tccacttgct cgagcctagt attttaactt 9840 cactatatac cttctttgat gacatatcct ttcaaatttt tggtcaagtt tttattgggg 9900 tgttgttact atggactgtg agagttcttt gtatattctg catatgattt ttttctcaca 9960 tttgtgtttt atgaatatgt tctcccaatg tgtggcgcct tttattttct taacgtgcca 10020 tgtgaagagc agaagtttaa ttttatgatg tccaaattat ctttttttct tttctttttt 100.80 agatcaaaat aggggtctat tttgattacc actgttattt tatctccatt tgattttcga 10140 tttttatttt tatttttcta atttcattgt aaatttttaa ttaaacccaa atattctagg 10200 ggaaagaggc aagataaaaa tagtctaact tgggcataåa ttttagagtc atattctctt 10260 gccgagaaag gaaactagct ctcttacatt gattgtttaa tttcagacgt cactacttta 10320 tgaggatgcc caaattatgg gctttaaaaa atatatatcc aaacaggggt tcagaaagaa 10380 taactaattt gtccacaaca acacaaaaaa tgattccacc ataagtttgc ccagtgacag 10440 ggtctatatt attttctata tatcaaattc tacaactggt tcttaaagct actgtacata 10500 acctaagtta aaatattagg tattagttga taagacattt tatcatctat gaaatgttgc 10560 ctgtt.gt.cat agttagagaa tcttttaaaa tatggagct.a ttttcataga ttaaactatg 10620 ccagttaaaa gttgggtaaa aagaactaca gaataatatt tatgtttatc gtgtaaggtt 10680 ttaaagcaaa ctccaagtca ttttcatcaa tgaaatcaat aaggttttgc aaatatatat 10740 gtatgaaaat actgatttaa aatgcaaata aggggagagt ttgagagaga gagagagacc 10800 aaatgatttt ataattctag taagtttata ggtttatggg gtttttacgt acttttctac 10860 ccaacttgtc tataagactt taatgaatca cttagaattt ttasaataat ttattattac 10920 tctgtacctg ttctttactc tgcaaatctt accttgccct tttgtctaaa agcaataaaa 10980 tctgacctgg tttatatcgt atcattgatt ttgttactta gcaagcacag tgatccatta 11040 ggcctatgta ggctcatggt ttatacaaca ctgccatctg ctgacagagt gtgacagtca 11100 cagtcagcaa cacgagacca ctttattttc atttttagtg tttatagaaa tatgaatata 11160 cacaaatagt ataatcaacc ctaagcttca caaattaaca ttttgctaat cttgtttcaa 11220 ctaccgcctc ccccctcatc caattactct gttctctcac ctcctcacac acagacactg 11280 gcagtatttt tcagccaatc attaatacgt tgccaactga taaggacttt taaaaaacaa 11340 ccaccattcc attatgattc ccagcataat tgagagtaat tccctaatat ccaataccca 11400 ttttctattc caatttcctt gattgtcttt aaactgtttt taccctaagt ttgcttaaat 11460 caaagtccag gtcetgttaa acatat.ggtt aagttttacc caaacccaaa taaataaata 11520 aataaataaa taaataacct attttttcca attccaggga atagtgaaag agggtaaatg 11580 ccattattta gaaacataaa tcacatcata ggactagaat tatcttgaag tcaaaattga 11640 agactgaaaa tggaaaagaa aggtatagac taaacttatt taaaaacttc aatgcagaac 11700 tctaagagaa gatattagaa agttgtacca gcattcatta ttcagtattc atcagtattc 11760 actcagctat atgtagttga aatctaacta gaggagcttg atcagataaa gagatacatt 11820 tttctcacca aggcggactc tggaggcagg tggttcagag ctagacagct gctgcaggac 11880 ccaggtcctt tccctgcctg ctcctc.cact ctagcttgtg actttcatcc tgcaagatgg 11940 gtgtttctgc caagttccag atagaagaag ataga'acaca aaggagaaat aagcagtggt 12000 gcctctgtcc atcaagcaaa atttttccag aaatgcacaa tagatttcag atgatgtctc 12060 aacagtccta actgcaaaga agctgaggaa ttagattttt ggctgggaca ctgttgccct 12120 qtaaaaaaaL tgggaLLcLg LLaLLaaaga ataagagqag ggaagaaaga ttgaaaactc 12180 ctatgcaata gtgaaaaaaa taagaaactc aataaaaaag tgggcatacc ttaaaaacag 12240 gcaattcaca acagatgaga ccccaatagc caataaacat ttttaaatgg tcaacctcat 12300 gagtgatcag aaaacacaaa tatgtatttt aaaccaaaaa taaaatacaa tgtattgacc 12360 atttgagtgg aaaaaaatta aaaagcctga taatatcaag tattggagag gat.gt.agagt 12420 gaggaaactc catggaggac ctatcattgc aaatgtggga atgaaactta atac.acgaat 12480 ttgaggccaa tttgtaaatt gaaaaatgcg cacaccctgc aaccaagtac cccttgcaat 12540 atttttgaaa agacaaaaac gttatgtaaa tggaatcatg caatatgtga cctttatact 12600 cagcataatg cccctcagat ccattgaagt catgtgtatc aacagctcac tatttttttt 12660 ttaatttttc ttagagacag agtctcactc tgtcacacag ggtcgagtgc agtggcgaga 12720 tcataactcc ctctagcagc ctcgaactcc tgggctcaag catcctcctg cctcagcctc 12780 ccaagtagct aggactacag gcatgggaca caacacacag ctaatttttt taaatttttt 12840 ttagagacat ggtctcacta tgttgcctac gctggtctca aactcctagg tcaagcgatt 12900 ctcccacctc tacttcacaa agtgctgtag gtatgtaggt atggattgta ggtatgaacc 12960 accgtgccca actcactact ttttattact aattattcca tgggatggat gtaacgcagt 13020 ttgttttacc attaatctat tgtaggacat tttgactgat tccagttttt ttttaataca 13080 aataaaacca ctatgaatag ttgtgtattg tatacgtttt tgtgctaagt tttcattttt 13140 ctgggataag ttttcatttc tttgggcttt tactgtatcc ttgatattat aatatgttac 13200 atcttcagtt ttattctatt caatatataa tcttttattt tccttgaaat ctcccatgga 13260 ttgtttagaa gtgtgttgtt ttgtttccaa gggtttggca tttttcccat tatttttcta 13320 ttatcgattt ccagtttgat tccaggtggt cagagaacac acttcatgtg atttcagttc 13380 tattaaattt gttgaggttt gttacatggc ccagtatatg gcaattttgg tatatgttcc 13440 atgagcactt gaaaagaatg cgaattctgc tggtgctggt tggagttttc cagcaatgtt 13500 gatttatgat cttactcatt gatggtggtg ttgagtttga tgtgttctta cgatggcagc 13560 tttaacattc ttgtcaggta attctaacgt ctctgtcatg tcagtattag cgcctcttaa 13620 ctgtctcatc aaagctgaga ttttcctggt tcccctggtt cctgttggga tgtgtggttt 13680 tcatttgaaa tctggacttt ggagtattgt gttatgaggc tttggatctc atttaaactc 13740 atctcagcga atttcctctc ttgccactca ggaaggagaa·gttgggtgtt tgaatggagc. 13800 agagccgtt.a ctgcctaaga attgttttac tgggcttccc ctttctttct cctttgacta 13850 gagagagcca gctttttatt agggctttat gtttttctgg gcctgttggt gtttctgggt 13920 tgacaaactt ctccagaacc aagtctggaa tggatgaggc aaaaagaaac cccgtggaat 13980 gcactgctgg gtcgctcctt gggtcccaat gttcctaact ggtctgcctt cttctctcca 14040 gcttccagag tcttcataag tttgctttac gtacaatgtc cggggttttt actttacttg 14100 agagaaatag ctaaaagtaa ttctactcca tctttcagga agcaaaagcc cccttgtgta 14160 tttttttaaa ctttcaaaaa caaaacaaaa ggcagctgca acagtaaaga agctagtaac 14220 acccttggtg ggaaattcaa gtccaaatac acattttaag tttggctagc cagtgagaac 14280 atcagaatag ttcaggtttt aaacaaattt atatttatga ttatgcatat actaaaagct 14340 gaaggcatct tatatttact aagcacctat tttgttcttg ttaaaaagac agaattccat 14400 tccctaggaa atttgacctg gcagctggag ctgatccacc tggccactag agcacagagc 14460 agggagagta gtagccctgc cccagccacc cctcaagaca ggattctttc tctgggaact 14520 gtaggtaaca ctaaatcgtt ctggaacaca acaacgaaag aagaaaggaa agagaaagaa 14580 agaaaggaag aaagagagag agaaggaagg aagggaggga gggaaggaag gaaggggaag 14640 ggaagggaat ggaagggaag gaaggaagga aaaggaagga agggagggag agagggagga 14700 aggaaggaaa ggaaaggaag gaaggaagaa ggaaagaaaa aaagaaagaa agaagaaaga 14760 aagaaagaca agaaagaaag aaagaaagaa agaaagggga aagaaaagaa agaggaaaga 14820 aagagaaaga aagaaaagaa agaaaggaaa gaaagagaaa gaaagaaaaa gaaagagaaa 14880 gaaagagaaa gacaagaaag aaaaaggaaa gaaaagaaag agaaagaaaa gaaagaaagg 14940 aaagaaagag aaagaaagaa aaagaaagaa agaaaagaag aaagagaaag aaagaaagaa 15000 aaagaaagaa agaaagaaag aaagaaaaag aaaaagaaag gagaaaatga cagcaattac 15060 ttttgcaaca acctaatata agttttttaa aagttaaata ttctgttcca tgcattgctg 15120 gataccttat aaataacagg gcatcctatg acctgaattt cccaaattat gagttgaggg 15180 tttgaactag ttttaaaaaa caaggaggcc aggcgcactg gctcatgcct gtaatcccag 15240 cactttggga ggctgaggca ggtggatcac gaggtcagga gctcgagacc agccttacca 15300 acatagtgaa acaccgcctc tactaaaaat acaaaaatta gccgggcgtg atggtgcgca 15360 cctgtaatct cagctactca gcaggctgag gcaggagaat cgcttgaacc cagaaggcgg 15420 aggttgcagt gagccaagat cacagcattg cactccagcc tgggcgacag agggagactc 15480 cgtcttcaaa aaaaaaaaaa aagacaagga atctgtaaaa caggcactgg aagtatatgc 15540 acttttattt tcattctatg ctatccgatg cctactgcta tttcccttca tatttaacct 15600 ccaacagctg cattttgctc cctccagacc acctgattgg agctcacgtg ctcccacaca 15660 gtacctccaa ccagagagag tcgagtccca cagaaaggcg taacaatcac cagtaatttt 15720 gcacttattt tacattgtgc cttgatacag agtactcaat gaatgctctC tgaatcatat 15780 ttaataaata tgtgtatttg ggattgtagc atattgcagc tacctggata tataatttaa 15840 ttagaaaaaa aattttgtgt ggctcaatca acaaacgact tttctctctc tctctttctc 15900 tttctccctc tctctctctt tcttctcagt tgatgttgct ggagttcagt gttgtgcaga 15960 tggcagtgac aaatgccatg ggcacatgag atatgataaa aggtccctga agaaggtgga 16020 gaaccagtta tcttatgaaa ttttccagag tgggtactgg atctctcctg tctggcacca 16080 tgctggcctc agcccaaggg gaatttcctt ccagagacag agggcagtga ctgaggtggg 16140 gagacagatc gtaacactga gacttacatg aggacaccaa acagaaaaaa ggtggcaagt 16200 atagaaaatt ctttcttctg gacagtcttc tctgttctaa cttcagcaaa attctccccc 16260 cagtggatgc tattgcacaa ccctacatat gctatgtttt ttcctataca cacttaccta 16320 tgataaaatg cattaattag tcacagtaag aggttaacaa caataactag taataaaata 16380 gaacaattca gtaaaataag agttacttga gcacaaacac taggatatca tgacagtcaa 16440 tctgatgacc aagagggcta ctaagcatct aaacaggagg gtaagtgtag acagcatgga 16500 gacgctggac aaagggatga ttcagtccca ggctggtatg gagcggaagg gcatgatatg 16560 tcatcacgct actaaggcac acaatttaaa atgagtaaat tcttatttct agaaatttct 16620 ttttaatatt ttrcagactac agttgcctac aggtaactga aaccccagaa agcaaaattg 16680 ttgataagga ggtactactg tacatcgtcc tttgaaccaa clttatcatt tgctagtata 16740 tacatatata cctacataca tacatataca catacctgca cacacctata tgtatacgta 16800 cacacacaca cacgcacaca cacacactca catctactaa tgttagaata agtttgctaa 16860 ataagatgca caacttgtta atgtcc-.aca gagcaataaa accataagca ttggggttat 16920 cttttctact agataaaaat ccattatcat tttcataaag ttttctttac attaacatct 16980 aacttttgca atctagtttt taatcatcat aaataggaag caaatgaact.gtttctctag 17040 tgaatcaaat atccttgaaa acatacatag tcatcttttt ggtttatttt tatttttaga 17100 taaattattt aaagttttaa ataatttaac attcacaata gtttgtgact gtatattttg 17160 acttggtcct tcaaacttaa tttgtacttt tatgtatcgt gcttacctca attttttatt 17220 cacttttcct aaactttgct ggattggttt attatttttg tctatttctt ttccttctag 17280 tggtttggga gggLltttta aatcccatta ctattgaatg cctattaact tgcccccttt 17340' ttctttcaat ctctattccc acggcctgaa gcatgagggc caagctgtct gtaaccagca 17400 gagagatgac ccaggtgtta ttccactctc cactgtccac ctatcaccat t.cccagcccg 17460 atagctctga agtarggctt ttc.tggggct ctgtggggaa aactagaact ggctgcttca 17520 aggacacctc ctgtttttgc aatggaaaaa atgtttctaa attccagttt ctctatgaat 17580 tcaatgacat ggtttaaatc tctgtggrgt tcttcaaagt tttttcttct aataggacct 17640 ctcatgattc tccaaccacg aaataaattc attatcattt ttatatttct tctgtcattg 17700 caaaggaggt tttgaaagag tggaggacgc gctaatgaac tcaaaaatcc acactattcc 17760 ttgtttccat ctgttgttca ttcattgttt ccattggcct gtccgcctcc tatcctcctt 17820 ctuagacttg gagctctagc ctcagccagg atagqqaaaa gagagatcag actgttactl 17880 tgtctatgta gaaaaggaag acataagaaa ctccattttg atctgtatcc tgaacaattg 17940 ttttgccttg agatgctgtt aatctgtaac tttagcccca accttgtgct cacagaaaca 18000 tgtgttgtat ggaatcaaga tttaagggat ctagggctgt gcagaatgtg ccttgttaac 18060 aacatgttta caggcagtat gcttggtaaa agtcatcgcc attctccatt ctcgattaac 18120 taggggcaca gtgcactgcg gaaagccgca gggacctctg cccaggaaaa ctgggtattg 18180 tccaaggttt ctcccoactg agacagcctg agatatggcc ttgcgggatg ggaaagatct 18240 gaccgtcccc cagcctgaca cccgtgaagg gtctgcgctg aggaggatta gtaaaagagg 18300 aaggcctctt gcggttgaga taagaggaag ccctctgtct cctgcatgcc cctgggaacg 18360 gcatgtctca gtgtaaaacc tgattgtaca ttcgttctat tctgagatag gagaaaaccg 18420 ctctgtggct ggaggcgaga tatgctggcg gcaatgctgc tctgttgttc tttaclacac 18480 tgaga-gttt gggtgagaga agcataaatc Lggcclacgt gcacatccag gcatagtacc 18540 ttccc^tgaa tttacttgtg acacagattc ctttgctcac atgttttctt gctgaccttc 18600 tccccactat caccctgttc tcctgccgca ttccccttgc tgaggtagtg aaaatagtaa 18660 tcaataaata ctgagggaac tcagagaccg gtgccagcgc gggtcctccg tatgctgagt 18720 gacggtccct tgggcccact gttccttctc tatactttgt ctctgtgtct tatttctttt 18780 ctcagtcrct cgtcccacct gacgagaaat acccacaggt gtggaggggc tggacacccc 18840 ttcgagccag gattatcagg gcatttgggg gtctgcaaaa ctaagcccca actcatcgat 18900 ttcacaactt catccagagc cagcctgaac agtagttgcc catgatttct atgccttaat 18960 acgagaagag aacatagggg ctgggtgcca agtaggtaga cagggagggc agggaactct 19020 aagacagagc ttgaggggct cattcctctt gcaaaatgaa acaaaaacca cagcactgaa 19080 tatgtaaatc tcggtggctg aacccctcct aggaLaglaa gccctgacac aattgctgct 19140 atcttctctt tctctcaagg aagLcaaaaa acacctgcag ccttactgtc cccttggaaa 19200 caagatgaac atctacattt tctaaagtgg gacaagaatc tctgttcata tttatgtccc 19260 atgcatttgc acgtggccgg acaaaggact ttgcttctgc cagcacatct gtcttcagat 19320 atgagaggaa acagacacaa cctggaggcg gcaaagaagc. agctctttct caagtgacct 19380 cctctatctc cctacttcct ggctaatggg gcagccttga tccttgggaa tccaggacag 19440 atatccactc gtgacaaact agctggaaga atgacaacca atcaggttcc aagcaccact 19500 ggatgtgaac cacagaattt cctcctctcc ttgtggaatg tcagcttacg tctgacaaaa 19560 aatgtaaaac tgagagagtt acaatcttaa ggaggagtca agctaaagca gaaagaatca 19620 cctactctgg actccagcat gactgctgag ctcaaatata tatagagaga gaaagaacca 19680 caaacttgaa gatggatatc agctacagac tttcctgagt caggtaggga aatggccatc 19740 cctcaaacct tgcaaaaggc aaacttatgc cattgtgtcc tctgacatac tgggtgatgt 19800 actgtatgtt actgatgtga ggggaacttc ctaaattggc tagtaaatta rgccaaataa 19860 aaagcaaaaa tgatatttct tgaaatgtta catctgagga acattgctaa aataatttat 1S920 cagtagtttt caggatgatt tatagatgtg cattgaagtg tgtacttgtg ctctctctct 1S980 cctctctctc tctctttctc tcctctctct cgctctttct ctccttgccc ccctccctcc 20040 ctgactttcc ttcctgtccc ctccacagca gtttatattt tttttctgat aatctaactt 20100 tgctgagggt tcaatgtaaa gcaccttcag tgatgagtta gttggaatgt tccccaagaa 20160 attctatttc cagcactctt ttacatgaaa tccaagaagc tctcagacta tcttactgac 20220 accttgcctt tcctcaacag atcaatctta tcaatgtcca tcacagatat tttgtagaac 20280 ggtggatcct ggcagagtct cacagatgct tctgagacaa catttgcttt caaaaaatga 20340 accacacaca tcctaaagat ctcagccact tcccatgttt cattttgtgt tacagcaaac 20400 atcacaacaa tcattcctac agatcaccac tgcatgtgat caataaaata gtttttgcaa 20460 caatggtact tatgataatc atcttttatt gtttacaaat actgctttac aatagttatt 20520 cggttgcact gttcatatta gatttccaat tagctcactt aggaacataa gtccctcgaa 20580 cagctcagtc atctttttca ttcctgtttc tatcccctac atctctttcc tttgcagacg 20640 actatctcct acactgaaac aggaaagctt ttaccttttt ggcatgcttg atttaaagat 20700 tatagaaaag tatttgacaa agaaaactca cacatgtgtg tacatatctt ttaaaaagtt 20760 atgtttatgc attgcacagg aatatcgaga atgctaatag gcaatgtcag agtttactgt 20820 ttttcaaaat tagtacagtt ttattatttc taaaaactat aaaatgaata tattcacatc 20880 accatacaga agagtaggag gagatggcat aaagtgtcat tgttcctcct ctgcaatccc 20940 aggagataac taccaagcac aatttatgtc ttttaaaatt cagcccgtat ttatatacat 21000 atatattcaa tgtagatggg atcatgatat ctcaccacac atactcttca gtgacctgca 21060 ttttcacaaa caccttccac gtaactatat agaagtctac gtcttcccct taatgtctgc 21120 tttgtgctac attgtaaagc tctagcacag tttaaccaaa ctcctattaa tgaggatttt 21180 agttattttt cactctttaa acaatatttc catgtgtagt cttatacata cgtctgtaca 21240 cacttatccc agtctaagga gttcctttta ccttccccca tcccagcatt ccctgtcacg 21300 cttgttgctt ccgttgagtg actttactcc tggagtataa tctgcgtata gttcagttaa 21360 aaacatggga tctgagttta ggtcacagct ctgccactta ctgccataag ccagttcctt 21420 gacctctctg ccctcaagtt tttgcaccta caaagtaggg gataatatta gttcctagtt 21480 catagagtct tgggaataat taaatgtgat gatccatgta caatgtctgg cacttagtaa 21540 gtgctcaata aatgtcaccc tttatgattg gtattgcgtg tatgtctgca gagaaaatca 21600 ctttgtgtcc cctttaaaaa aggactatgc ccttggtcag ctattttgca cattaaattt 21660 cacttgccaa tattaactct ccacctctaa cttgatccct ctccttcctc atcttctggt 21720 gagaccaaat gctaattctg ctattcaagg caactagcaa agctgccagt gacagaatca 21780 aataaaccta cccctaatct ttagaattgt agttatgatt tctgttgtaa aagttactgt 21840 tgtggcagtc agtattagtc tttggtctat gatagcatct ctgatctatt attgaytttc 21900 aattakgtat ttttttttat ttattctgaa aatgtttgtt aagcatttgc taagtaaaga 21960 tactggackg agcctcccaa atacagggca aataaaacat caaacagctt ataatttaga 22020 agggtagaag agaatctgaa agcaggtaaa aataaacagg cactcggctg ggcgcggtgg 22080 ctcacgcctg taatcccagc actttgggag gccgaggtgg gcggatcacg aggtcaggag 22140 atcgagacca tcctggctaa cacggtgaaa ccccgtctct actaaaaata caaaaaatta 22200 gcgaggcgtg gtggcgggcg cctttagtcc cagctagtcg ggaggctgag gcaggagaat 22260 ggtgtgaacc cgggaggcgg agcttgcagt gagccaagat cgcaccactg cactccagcc 22320 tgggygacag agcgagactc cgtctcaaaa aaaataaata aataaaataa aaaataatta 22380 ggtactctag gcccagtgac ctgtctctgt actctgtaaa ttcaggtcac ctgctcaggg 22440 ctaatctgag agaaggtctc tcttcagttg aattttgaaa gacaattagc agttcacaag 22500 ctaacccagg tggacaaaga tgttcccaag cagagggagt gcttgtgaaa gctggaggcc 22560 atagaaaaac tctaaggagt gtagggaggt gggagtaatg tatggaaggg gtggagatgg 22620 aaggttaaga gagatacaag gctgcaaaaa tggagctgga ctcaaaagaa aatactgaaa 22680 aggtcttcag tgttgttgat gagattacta tggaaacact atggaacact gggactccat 22740 ggcagctcca aagatggcat gcgcctggtc cagctcagta agagctgagc tcttcctgtg 22800

<210> 101 <211 > 154 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 101 tctggcaaca cggcctccct gaccgtctct gggctccagg ctgaggatga ggctgattat 60 tactgcagct catatgcagg cagcaacaat ttaagtcttc ggaactggga ccaaggtcac 120 cgtcctaggt cagcccaagt ccactcccac tete 154

<210> 102 <211 > 156 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 102 tcagggacaa tggccacctt gaetateagt ggggcccagg tggaggatga agctgactac 60 taetgttaet caacagacag cagtggtaat cattatgtct teggaaetgg gaccaaggtc 120 accgtcctag gtcagcccaa gtccactccc actctc 156

<210> 103 <211 > 150 <212> DNA <213> Artificial Sequence <220> <223> synthetic <900> 103 tctgggaaca cagccactct gaccatcagc gggacccagg ctatggatga ggctgactat 60 tactgtcagg cgtgggacag cagcactgcc gtcttcggaa ctgggaccaa ggtcaccgtc 120 ctaggtcagc ccaagtccac tcccactctc 150

<210> 104 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 104 aggtggaaac acggtgagag t 21

<210> 105 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400>105 ccactcgggg aaaagttgga a 21

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • WQ0026373A iODOSl • VVQ2010039900A [00061 • US6998514B [01651 • US7435S713 [0165] . US65862513 Γ019β1 • US-37742798 [02241 • US7294754B 102251 • US65965413 Γ0243] • WQ61357314A [0250] • WQ61357317A [0250]

Non-patent literature cited in the description • VALENZUELA et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysisNature Biotech., 2003, vol. 21,6652-659 £0196] • POUEYMIROU et al.FO generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analysesNature Biotech., 2007, vol. 25, 191-99 [0225]

Claims (17)

Translated fromEnglish

1. Mus, der omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joigning- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et ikke-omarrangeret humant lambda variabelt (hVA) og et ikke-omarrangeret humant lambda joining-(hJA) gensegment, hvor musen omfatter en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og erstatning, fuldstændigt eller delvist, af VA-JA-gensegmenterfra den anden VA-JA-CA-genklynge fra allelen med de humane VA-og JA-gensegmenter, der er operativt forbundet til en intakt muselambdakon-stant- (CA) region fra den anden VA-JA-CA-genklynge, således at musen udtrykker en letkæde afledt fra et hVA-, et hJA- og et muse CA-gen, hvor de endogene enhancere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeallelen.A mouse comprising: (a) replacing one or more heavy chain (Vh), diversity (Dh), and joining (Jh) gene segments with one or more human Vh, Dh and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a non-rearranged human lambda variable (hVA) and a non-rearranged human lambda joining (hJA) gene segment, wherein the mouse comprises a deletion of a first VA-JA-CA gene cluster from the endogenous mouse λ light chain allele and replacement, in whole or in part, of the VA-JA gene segment from the other VA-JA-CA gene cluster from the allele with the human VA and JA gene segments operatively linked to an intact mouse lambda constant (CA) region from the second VA-JA-CA gene cluster such that the mouse expresses a light chain derived from an hVA, a hJA and a mouse CA gene, where the endogenous enhancers are Enh 2.4, mouse lambda 3 'Enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.2. Mus ifølge krav 1, hvor den endogene A- (lambda) letkædeallel omfatter et antal af humane VA-gensegmenter.The mouse of claim 1, wherein the endogenous A (lambda) light chain allele comprises a plurality of human VA gene segments.3. Mus ifølge et hvilket som helst af de foregående krav, hvor den endogene A-(lambda) letkædeallel omfatter: (a) mindst 12 omarrangerede humane VA-gensegmenter eller (b) 13 til 28 omarrangerede humane VA-gensegmenter.The mouse of any preceding claim, wherein the endogenous A (lambda) light chain allele comprises: (a) at least 12 rearranged human VA gene segments or (b) 13 to 28 rearranged human VA gene segments.4. Mus ifølge krav 3, hvor de humane VA-gensegmenter indbefatter (a) hVA3-1, hVA4-3, hVA2-8, hVA3-8, hVA3-10, hVA2-11 og hVA3-12 eller (b) hVA2-14, hVA3-16, hVA2-18, hVA3-19, hVA3-21, hVA3-22, hVA2-23, hVA3-25 og hVA3-27.The mouse of claim 3, wherein the human VA gene segments include (a) hVA3-1, hVA4-3, hVA2-8, hVA3-8, hVA3-10, hVA2-11 and hVA3-12, or (b) hVA2- 14, hVA3-16, hVA2-18, hVA3-19, hVA3-21, hVA3-22, hVA2-23, hVA3-25 and hVA3-27.5. Mus ifølge krav 1, hvor den endogene A- (lambda) letkædeallel omfatter et omarrangeret JA-gensegment, der er hJA1.The mouse of claim 1, wherein the endogenous A (lambda) light chain allele comprises a rearranged JA gene segment which is hJA1.6. Mus ifølge krav 1, hvor den endogene A- (lambda) letkædeallel omfatter fire omarrangerede humane JA-gensegmenter.The mouse of claim 1, wherein the endogenous A (lambda) light chain allele comprises four rearranged human JA gene segments.7. Mus ifølge krav 6, hvor de fire omarrangerede humane JA-gensegmenter er JA1,JA2, JA3 og JA7.The mouse of claim 6, wherein the four rearranged human JA gene segments are JA1, JA2, JA3 and JA7.8. Mus ifølge krav 1, hvor den endogene A- (lambda) letkædeallel mangler et endogent VA-gensegment.The mouse of claim 1, wherein the endogenous A (lambda) light chain allele lacks an endogenous VA gene segment.9. Mus ifølge et hvilket som helst af kravene 1 til 3, 5 og 8, hvor den endogene K- (kappa) letkædeallel er deleteret.A mouse according to any one of claims 1 to 3, 5 and 8, wherein the endogenous K- (kappa) light chain allele is deleted.10. Mus ifølge krav 1, hvor den endogene muse λ-letkædeallel omfatter: (a) en tilstødende sekvens fra det humane λ-letkædelocus, der spænder fra VA3-12 til VA3-1; (b) en tilstødende sekvens fra det humane λ-letkædelocus, der spænder fra VA3-29 til VA3-1; eller (c) en tilstødende sekvens fra det humane λ-letkædelocus, der spænder fra VA3-29 til VA3-1 og en tilstødende sekvens fra det humane λ-letkædelocus, der spænder fra VA5-52 til VA1-40.The mouse of claim 1, wherein the endogenous mouse λ light chain allele comprises: (a) an adjacent sequence of the human λ light chain locus ranging from VA3-12 to VA3-1; (b) an adjacent sequence of the human λ light chain locus ranging from VA3-29 to VA3-1; or (c) an adjacent sequence from the human λ light chain locus ranging from VA3-29 to VA3-1 and an adjacent sequence from the human λ light chain locus ranging from VA5-52 to VA1-40.11. Isoleret celle, der udtrykker en letkæde afledt fra et hVA-, et hJA- og et muse CA-gen, hvor cellen er fra, eller opnået fra, musen ifølge et hvilket som helst af de foregående krav, og hvor cellen omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joigning- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et omarrangeret humant lambda variabelt (hVA) og et omarrangeret humant lambda joining- (hJA) gensegment, og en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og erstatning, fuldstændigt eller delvist, af VA-JA, gensegmenter fra den anden VA-JA-CA-genklynge af allelen med de humane VA- og JA-gensegmenter, der er operativt forbundet til et intakt CA-muse-konstant-region- gen af den anden VA-JA-CA-genklynge, og hvor de endogene enhancere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeal-lelen.An isolated cell expressing a light chain derived from an hVA, a hJA and a mouse CA gene, wherein the cell is from, or obtained from, the mouse of any of the preceding claims, wherein the cell comprises: (a) replacing one or more heavy chain (Vh), diversity (Dh), and joining (Jh) gene segments with one or more human Vh, Dh and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster from the endogenous mouse λ light chain allele and replacement, in whole or in part, of VA-JA, gene segments of the second VA-JA-CA gene cluster of the allele with the human VA and JA gene segments operatively linked to an intact CA mouse constant region of the second VA-JA-CA gene cluster, and where the endogenous enhancers Enh 2.4, mouse lambda 3 'enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.12. Isoleret celle ifølge krav 11, hvor cellen er en B-celle.An isolated cell according to claim 11, wherein the cell is a B cell.13. Isoleret embryonal musestamcelle (ES), der omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joigning- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et ikke-omarrangeret humant lambda variabelt (hVA) og et ikke-omarrangeret humant lambda joining-(hJA) gensegment, og en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og en erstatning, fuldstændigt eller delvist, af VA-JA-gensegmenterne af den anden VA-JA-CA-genklynge af allelen med de humane VA- og JA-gensegmenter, der er operativt forbundet til et intakt muse CA-konstant-region-gen fra den anden VA-JA-CA-genklynge, hvor de endogene enhancere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeallelen.An isolated embryonic mouse stem cell (ES) comprising: (a) replacing one or more heavy chain variables (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh and Jh gene segments at an endogenous mouse tongue chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a non-rearranged human lambda variable (hVA) and a non-rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster. from the endogenous mouse λ light chain allele and a replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of the allele with the human VA and JA gene segments operatively linked to an intact mouse CA constant region gene from the second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3 'enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.14. Museembryo, der omfatter, er lavet fra, eller opnået fra, muse ES-cellen ifølge krav 13, hvor museembryoet omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joining- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et ikke-omarrangeret humant lambda variabelt (hVA) og et ikke-omarrangeret humant lambda joining-(hJA) gensegment, og en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og en erstatning, fuldstændigt eller delvist, af VA-JA-gensegmenterne fra den anden VA-JA-CA-genklynge af allelen med de humane VA- og JA-gensegmenter, der er operativt forbundet med et intakt muse CA-kon- stant-region-gen fra den anden VA-JA-CA-genklynge, hvor de endogene enhan-cere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeallelen.The mouse embryo comprising, is made from, or obtained from, the mouse ES cell of claim 13, wherein the mouse embryo comprises: (a) a replacement of one or more heavy chain variables (Vh), diversity (Dh) and joining- ( Jh) gene segments with one or more human Vh, Dh and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a non-rearranged human lambda variable (hVA) and a non-rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster. from the endogenous mouse λ light chain allele and a replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of the allele with the human VA and JA gene segments operatively associated with an intact mouse CA constant region gene from the second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3 'enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.15. Anvendelse af B-cellen ifølge krav 12 til fremstilling af et hybridom, der udtrykker en letkæde afledt fra et hVA-, et hJA- og et muse CA-gen, hvor hybridomet er fra, eller opnået fra, musen ifølge et hvilket som helst af kravene 1 til 10, hvor cellen omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joigning- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et omarrangeret humant lambda variabelt (hVA) og et omarrangeret humant lambda joining- (hJA) gensegment, og en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og erstatning, fuldstændigt eller delvist, af VA-JA-genseg-menterne af den anden VA-JA-CA-genklynge af allelen med de humane VA- og JA-gensegmenter, der er operativt forbundet med et intakt, muse CA-konstant-region-gen fra den anden VA-JA-CA-genklynge, og hvor de endogene enhancere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeallellen.Use of the B cell of claim 12 for the preparation of a hybridoma expressing a light chain derived from an hVA, a hJA and a mouse CA gene, wherein the hybridoma is from, or obtained from, the mouse of any of the any of claims 1 to 10, wherein the cell comprises: (a) replacing one or more heavy chain variables (Vh), diversity (Dh) and joining (Jh) gene segments with one or more human Vh, Dh and Jh gene segments at a endogenous mouse tongue chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster from the endogenous mouse λ light chain allele and replacement, in whole or in part, of the VA-JA gene segments of the second VA-JA-CA gene cluster of the allele with the human VA and JA gene segments operatively associated with an intact mouse CA constant region gene from the second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3 'enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.16. Hybridom, der udtrykker en letkæde afledt fra et hVA-, et hJA- og et muse CA-gen, hvor hybridomet er fra, eller opnået fra, musen ifølge et hvilket som helst af kravene 1 til 10, hvor cellen omfatter: (a) en erstatning af ét eller flere tungkædevariable (Vh), diversitets- (Dh) og joigning- (Jh) gensegmenter med ét eller flere humane Vh, Dh og Jh gensegmenter ved et endogent musetungkædeimmunoglobulinlocus; og (b) en endogen A- (lambda) letkædeallel omfattende et omarrangeret humant lambda variabelt (hVA) og et omarrangeret humant lambda joining- (hJA) gensegment, og en deletion af en første VA-JA-CA-genklynge fra den endogene muse λ-letkædeallel og erstatning, fuldstændigt eller delvist, af VA-JA-genseg-menterne fra den anden VA-JA-CA-genklynge af allelen med de humane VA- og JA-gensegmenter, der er operativt forbundet med en intakt muse CA-konstant-region-gen fra den anden VA-JA-CA-genklynge, og hvor de endogene enhancere Enh 2.4, muselambda 3'-enhancer (Enh) og Enh 3.1 opretholdes intakte i A-letkædeallelen.A hybridoma expressing a light chain derived from an hVA, a hJA, and a mouse CA gene, wherein the hybridoma is from, or obtained from, the mouse of any one of claims 1 to 10, wherein the cell comprises: ( a) replacing one or more heavy chain (Vh), diversity (Dh), and joining (Jh) gene segments with one or more human Vh, Dh and Jh gene segments at an endogenous mouse heavy chain immunoglobulin locus; and (b) an endogenous A (lambda) light chain allele comprising a rearranged human lambda variable (hVA) and a rearranged human lambda joining (hJA) gene segment, and a deletion of a first VA-JA-CA gene cluster from the endogenous mouse λ light chain allele and replacement, in whole or in part, of the VA-JA gene segments from the second VA-JA-CA gene cluster of the allele with the human VA and JA gene segments operatively associated with an intact mouse CA constant region gene from the second VA-JA-CA gene cluster, where the endogenous enhancers Enh 2.4, mouse lambda 3 'enhancer (Enh) and Enh 3.1 are maintained intact in the A light chain allele.17. Fremgangsmåde til fremstilling af et antistof hos en mus, der omfatter: (a) eksponering af en mus ifølge krav 1 for et antigen; (b) muliggøre, at musen udvikler et immunrespons over for antigenet; og (c) isolering fra musen af (b) et antistof, der specifikt genkender antigenet, eller isolering fra musen af (b) en celle, der omfatter et immunoglobulindomæne, der specifikt genkender antigenet, eller identificering i musen af (b) en nukleinsy-resekvens, der koder for et variabelt tung- og/eller letkædedomæne, der binder antigenet, hvor antistoffet omfatter en letkæde afledt fra et hVA-, et hJA- og et muse CA-gen.A method of producing an antibody in a mouse comprising: (a) exposing a mouse according to claim 1 to an antigen; (b) enabling the mouse to develop an immune response to the antigen; and (c) isolating from the mouse of (b) an antibody that specifically recognizes the antigen, or isolating from the mouse of (b) a cell comprising an immunoglobulin domain that specifically recognizes the antigen, or identifying in the mouse with (b) a nucleic acid sequence encoding a variable heavy and / or light chain domain that binds the antigen, wherein the antibody comprises a light chain derived from an hVA, an hJA and a mouse CA gene.